Herpes simplex virus (HSV) vaccine

Abstract

The present invention relates to a herpes simplex virus (HSV) vaccine. The herpes simplex virus vaccine comprises one or more of the following nucleic acids containing polynucleotides that encode HSV glycoproteins or immunogenic fragments thereof: a nucleic acid containing a polynucleotide that encodes HSV glycoprotein D or an immunogenic fragment thereof, a nucleic acid containing a polynucleotide that encodes HSV glycoprotein C or an immunogenic fragment thereof, and a nucleic acid containing a polynucleotide that encodes HSV glycoprotein E or an immunogenic fragment thereof.

Description

Herpes simplex virus (HSV) vaccine TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a herpes simplex virus (HSV) vaccine. BACKGROUND

[0002] Herpes simplex virus (HSV) belongs to the alpha subfamily of herpesviruses, and is a double-stranded DNA virus with an envelope structure. Herpes simplex virus includes two serotypes: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).

[0003] HSV can cause two different forms of infection: primary infection and latent infection. Primary infection refers to the first proliferation of HSV in epithelial cells and their surrounding tissues in the peripheral mucosa after the virus contacts the host, and the lesion formed is a primary infection lesion. After HSV infects the peripheral tissue, a part of the virus contacts the axon of the peripheral neuron (mainly sensory neurons), and moves to the cell body along the axon to establish latent infection. HSV-1 and HSV-2 mainly latent in the trigeminal ganglion and sacral ganglion, respectively. HSV establishes latent infection in nerve cells, and takes advantage of the low metabolic characteristics of nerve cells, so that the antigen of HSV is not easily presented to immune cells, thereby escaping immune recognition. When the host's immunity decreases or is stimulated by stress factors, the virus will re-replicate in the ganglion, and then move to the skin mucosa surface through the neuron axon to activate and form recurrent infection.

[0004] HSV-1 infection can cause oral and lip inflammation, and can induce conjunctivitis to cause blindness when the infection is severe. It can also invade nerve cells to damage the nervous system and cause encephalitis. HSV-2 mainly causes genital herpes, and once infected, the patient will carry the virus for life and have periodic genital herpes lesions.

[0005] In recent years, epidemiological investigations have shown that the incidence of diseases such as oral and lip herpes, pharyngitis, and genital herpes caused by HSV is at a high level. However, the unique infection characteristics and immune escape mechanism of HSV enable it to both evade host immunity and drug treatment, and to massively infect humans. Therefore, it is particularly important to develop an HSV vaccine. SUMMARY

[0006] The present disclosure provides a nucleic acid composition comprising one or more of the following nucleic acids containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof:

[0007] (1) a nucleic acid comprising a polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof;

[0008] (2) a nucleic acid comprising a polynucleotide encoding HSV glycoprotein C or an immunogenic fragment thereof; and

[0009] (3) a nucleic acid comprising a polynucleotide encoding HSV glycoprotein E or an immunogenic fragment thereof.

[0010] In some embodiments, the nucleic acid composition comprises each independently a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof, the HSV-2 glycoproteins or immunogenic fragments thereof further linked to a heterologous signal peptide, respectively.

[0011] In some embodiments, the heterologous signal peptide comprises one or both of the following: an IgE signal peptide and an IgG kappa signal peptide.

[0012] In some embodiments, the heterologous signal peptide is an IgG kappa signal peptide.

[0013] In some embodiments, the amino acid sequence of the IgG kappa signal peptide is set forth in SEQ ID NO: 31.

[0014] In some embodiments, the nucleotide sequence of the polynucleotide encoding the IgG kappa signal peptide is set forth in SEQ ID NO: 32.

[0015] In some embodiments, the HSV glycoprotein D immunogenic fragment comprises a truncated HSV-2 glycoprotein D retaining positions 26-393, 26-363, or 26-331 of HSV-2 glycoprotein D.

[0016] In some embodiments, the HSV glycoprotein D immunogenic fragment comprises one of the following:

[0017] (1) a truncated HSV-2 glycoprotein D having an amino acid sequence set forth in SEQ ID NO: 1, 4, or 7; and

[0018] (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 1, 4, or 7.

[0019] In some embodiments, the HSV glycoprotein C immunogenic fragment comprises a truncated HSV-2 glycoprotein C that retains positions 28-480, 28-468, or 27-426 of HSV-2 glycoprotein C.

[0020] In some embodiments, the HSV glycoprotein C immunogenic fragment comprises one of:

[0021] (1) a truncated HSV-2 glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 2, 5, or 8; and

[0022] (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95% identical and less than 100% identical to an amino acid sequence as set forth in SEQ ID NO: 2, 5, or 8.

[0023] In some embodiments, the HSV glycoprotein E immunogenic fragment comprises a truncated HSV-2 glycoprotein E that retains positions 21-545, 24-548, 24-442, or 24-405 of HSV-2 glycoprotein E.

[0024] In some embodiments, the HSV glycoprotein E immunogenic fragment comprises one of:

[0025] (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 3, 6, 9, or 42; and

[0026] (2) a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95% identical and less than 100% identical to an amino acid sequence as set forth in SEQ ID NO: 3, 6, 9, or 42.

[0027] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached has an amino acid sequence as set forth in SEQ ID NO: 33; the HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached has an amino acid sequence as set forth in SEQ ID NO: 34; and the HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached has an amino acid sequence as set forth in SEQ ID NO: 35.

[0028] In some embodiments, the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 19, and that encodes an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 33, or its corresponding RNA.

[0029] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 20, and that encodes an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 34, or its corresponding RNA.

[0030] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 21, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 21, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 35, or its corresponding RNA.

[0031] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 33; the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 34; and the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 44.

[0032] In some embodiments, the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 19, and that encodes an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 33, or its corresponding RNA.

[0033] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 20, and that encodes an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 34, or its corresponding RNA.

[0034] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 45, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 45, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 44, or its corresponding RNA.

[0035] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 36; the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 37; and the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 38.

[0036] In some embodiments, the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 22, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 22, and that encodes an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 36, or its corresponding RNA.

[0037] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 23, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 23, and that encodes an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 37, or its corresponding RNA.

[0038] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 24, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 24, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 38, or its corresponding RNA.

[0039] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 39; the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 40; and the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 41.

[0040] In some embodiments, the nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 25, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 25, and that encodes an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 39, or its corresponding RNA.

[0041] The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 26, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 26, and that encodes an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 40, or its corresponding RNA.

[0042] The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 27, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 27, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 41, or its corresponding RNA.

[0043] In some embodiments, the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or immunogenic fragment thereof further comprises at least one of a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0044] In some embodiments, the 5'-UTR corresponds to a DNA sequence as set forth in SEQ ID NO: 28.

[0045] In some embodiments, the 3'-UTR corresponds to a DNA sequence set forth in SEQ ID NO: 29.

[0046] In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides in succession. In some embodiments, the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail corresponds to a DNA sequence set forth in SEQ ID NO: 30.

[0047] In some embodiments, the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof is RNA.

[0048] In some embodiments, the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof is mRNA.

[0049] In some embodiments, the mRNA comprises a 5'-cap structure.

[0050] In some embodiments, the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof is DNA.

[0051] In some embodiments, the DNA is capable of being transcribed into RNA.

[0052] In some embodiments, the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof contains modified nucleotides.

[0053] In some embodiments, the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof contains modified nucleosides.

[0054] In some embodiments, the modified nucleosides comprise at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0055] The present disclosure also provides a protein encoded by the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof in the nucleic acid composition.

[0056] The present disclosure also provides a non-natural nucleic acid, which is the nucleic acid containing the polynucleotide encoding the HSV glycoprotein or immunogenic fragment thereof in the nucleic acid composition.

[0057] The present disclosure also provides a pharmaceutical composition comprising the nucleic acid composition, the protein, or the unnatural nucleic acid.

[0058] In some embodiments, the nucleic acid composition or the unnatural nucleic acid is formulated in a delivery vehicle.

[0059] In some embodiments, the delivery vehicle is a lipid nanoparticle in which the nucleic acid composition or the unnatural nucleic acid is encapsulated.

[0060] In some embodiments, the pharmaceutical composition is a vaccine.

[0061] In some embodiments, the pharmaceutical composition is an mRNA vaccine.

[0062] The present disclosure also provides use of the nucleic acid composition, the protein, the unnatural nucleic acid, or the pharmaceutical composition in the preparation of a medicament for preventing or treating HSV infection.

[0063] In some embodiments, the HSV infection comprises HSV-2 infection.

[0064] In some embodiments, the HSV infection is a primary HSV infection or reactivation of latent HSV infection.

[0065] In some embodiments, the HSV infection comprises HSV neonatal infection or genital HSV infection. BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 is the titer of HSV-2 neutralizing antibodies in serum of mice immunized with mRNA vaccines; A1, A2, A3, A4 in FIG. 1 respectively refer to experimental groups injected with vaccines A1, A2, A3, A4, and “blank LNP” refers to an experimental group injected with blank LNP, and the other figures are the same.

[0067] FIGS. 2A-2C are ELISpot results of IFN-γ of mice immunized with mRNA vaccines;

[0068] FIGS. 3A-3C are ICS results of IFN-γ of CD4+ T cells of mice immunized with mRNA vaccines;

[0069] FIGS. 4A-4C are ICS results of IFN-γ of CD8+ T cells of mice immunized with mRNA vaccines;

[0070] FIGS. 5A-5C are the effect of mRNA vaccines on the body weight of guinea pigs;

[0071] FIG. 6 is the effect of mRNA vaccines on the survival rate of guinea pigs;

[0072] Figure 7 shows the effect of mRNA vaccine on body temperature of guinea pigs;

[0073] Figure 8 shows the effect of mRNA vaccine on herpes incidence of guinea pigs;

[0074] Figure 9 shows the effect of mRNA vaccine on herpes score of guinea pigs;

[0075] Figure 10 shows the effect of mRNA vaccine on skin score of guinea pigs;

[0076] Figure 11 shows the effect of mRNA vaccine on clinical score of guinea pigs;

[0077] Figure 12 shows the titer of neutralizing antibodies against HSV-2 in serum of mRNA vaccine immunized guinea pigs;

[0078] Figure 13 shows the HSV viral titer in vaginal lavage of guinea pigs after challenge;

[0079] Figure 14 shows the HSV viral copy number in dorsal root ganglion of guinea pigs after challenge.

[0080] DETAILED DESCRIPTION

[0081] I. DEFINITIONS

[0082] All patents, patent applications, scientific publications, manufacturer's specifications and descriptions, and the like, cited herein are hereby incorporated herein by reference, whether or not specifically done so within the text of this disclosure. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0083] Unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be understood by one of ordinary skill in the art. Also, the terms of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and the like, used herein are terms that are well understood in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Also, for better understanding of the present application, the following provides definitions and explanations of relevant terms.

[0084] As used herein, the expressions "comprising", "containing", "including" and "having" are inclusive or open-ended and specify the presence of stated elements, steps, or components but do not preclude the presence or addition of one or more other, unstated elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of does not exclude the presence or addition of additional optional elements, steps, or components, provided that the presence of such additional optional elements, steps, or components do not materially affect the basic and novel properties of the claimed subject matter. It is to be understood that the expressions "consisting essentially of and "consisting of" encompass the meaning of the expression "comprising".

[0085] As used herein in the context of describing the application, particularly in the context of the claims, the singular forms "a," "an," and "the" and "said" and similar reference(s) are to be construed as including plural forms. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, or more. The terms "at least one" or "one or more" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0086] Numerical ranges recited herein are inclusive of the numbers within the range. For example, the range "1 to 10" is inclusive of the endpoints 1 and 10, as well as any single number between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and sub-ranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). This principle applies to ranges recited only with a minimum value or a maximum value.

[0087] As used herein, the terms "and / or", "any combination thereof and their grammatical equivalents are used interchangeably. These terms can express, in particular, any combination. For example, the following phrases "A, B and / or C" or "A, B, C or any combination thereof can mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C".

[0088] As used herein, the term "naturally occurring" or "naturally present" refers to the fact that a substance can be found in nature. For example, a peptide, amino acid, protein or nucleic acid that is present in an organism (including a virus) and that can be isolated from a source in nature and that has not been artificially modified in an experiment is naturally occurring.

[0089] As used herein, the term "non-naturally occurring" or "non-native" when used in reference to a nucleic acid herein is intended to mean that the nucleic acid is not found in nature. For example, a non-naturally occurring nucleic acid encoding a viral peptide segment or protein has at least one genetic alteration or chemical modification that is not normally found in a wild-type strain of the referenced virus. Genetic alterations include, for example, the introduction of an expressible nucleic acid sequence encoding a peptide segment or polypeptide heterologous to the referenced virus, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and / or other functional disruptions of the viral genetic material. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0090] Unless otherwise indicated, all methods described herein can be performed in any suitable order.

[0091] As used herein, the term "wild-type" indicates that the sequence is naturally occurring and has not been modified by man, including naturally occurring mutants.

[0092] As used herein, the term "% identity" or "% identity" with respect to the term refers to the percentage of nucleotides or amino acids that are the same in the optimal alignment of the sequences to be compared, the differences between the two sequences can be distributed over local regions (segments) or the entire length of the sequences to be compared. The identity between two sequences is usually determined after optimal alignment of a segment or "comparison window". The optimal alignment can be performed manually or with the aid of algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482, and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. The percentage identity of two sequences can be determined, for example, using the BLASTN or BLASTP algorithm publicly available on the website of the National Center for Biotechnology Information (NCBI).

[0093] "Percent identity" or "% identity" can be obtained by determining the number of positions in the two sequences which correspond to identical positions, dividing that number by the number of positions in the reference sequence (i.e., the length of the reference sequence), and multiplying the result by 100. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the region gives the degree of identity. In some embodiments, the entire length of the reference sequence gives the degree of identity. Alignment for determining sequence identity can be performed using tools known in the art, preferably with optimal sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0094] As used herein, "nucleotides" include deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotides" are the constituent materials of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group, while "deoxyribonucleotides" are the constituent materials of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydrogen instead of a hydroxyl group at the 2' position of the β-D-ribofuranosyl group, which is the main chemical component of chromosomes.

[0095] "Nucleotides" are generally referred to by a single letter representing the base therein, "A" or "A nucleotide" refers to an adenine deoxyribonucleotide or an adenine ribonucleotide containing adenine, "C" or "C nucleotide" refers to a cytosine deoxyribonucleotide or a cytosine ribonucleotide containing cytosine, "G" or "G nucleotide" refers to a guanine deoxyribonucleotide or a guanine ribonucleotide containing guanine, "U" or "U nucleotide" refers to a uracil ribonucleotide containing uracil, and "T" or "T nucleotide" refers to a thymine deoxyribonucleotide containing thymine.

[0096] As used herein, the term "nucleic acid" generally refers to any compound comprising a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA) or a combination thereof. In addition, nucleic acid herein also includes derivatives of nucleic acid. The term "derivative of nucleic acid" includes chemical derivatization of nucleic acid on the base, sugar, or phosphate of the nucleotide, as well as nucleic acid containing non-natural nucleotides and nucleotide analogs. Furthermore, in this document, nucleic acid can be in the form of a single-stranded or double-stranded linear or covalently closed circular molecule.

[0097] "Polynucleotide sequence", "nucleic acid sequence", and "nucleotide sequence" can be used interchangeably to indicate the ordering of nucleotides in a polynucleotide. It is understood by those skilled in the art that a DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidylate in the DNA coding strand sequence corresponding to the uridylate in the encoded RNA sequence. DNA corresponding RNA refers to the polynucleotide after all T in the DNA is replaced by U.

[0098] A polynucleotide can comprise one segment or multiple segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). For example, a polynucleotide can comprise a segment encoding a polypeptide of interest. In particular embodiments, a polynucleotide can comprise a segment encoding a polypeptide of interest and regulatory segments (including but not limited to segments for transcriptional regulation and translational regulation). In an embodiment, the regulatory segments comprise polynucleotides corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0099] As used herein, the term "promoter" refers to a polynucleotide located at the 5' end upstream of the coding region of a gene, which contains conserved sequences required for specific binding and transcription initiation of RNA polymerase, and can activate RNA polymerase to accurately bind to the template DNA and have specificity of transcription initiation. The promoter can be derived from viruses, bacteria, fungi, plants, insects, and animals. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter, or an SV 40 late promoter and a CMV IE promoter.

[0100] As used herein, the term “5’ untranslated region” or “5’-UTR” can be a sequence of RNA in an mRNA that is upstream of a coding sequence and is not translated into protein. The 5’-UTR in a gene typically begins at the transcription start site and ends at a nucleotide upstream of the translation initiation codon of the coding sequence. The 5’-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5’-terminal oligopyrimidine tract, and a translation initiation signal such as a Kozak sequence. The mRNA can be post-transcriptionally modified by the addition of a 5’ cap. Thus, the 5’-UTR in a mature mRNA can also refer to the sequence of RNA between the 5’ cap and the start codon.

[0101] As used herein, the term “3’ untranslated region” or “3’-UTR” can be a sequence of RNA in an mRNA that is downstream of a coding sequence and is not translated into protein. The 3’-UTR in an mRNA is located between the stop codon of the coding sequence and the poly(A) sequence, for example, beginning at a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence.

[0102] As used herein, the terms “polyadenosine,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably, and a naturally occurring poly(A) sequence typically consists of adenosine ribonucleotides. The poly(A) sequence is typically located at the 3’ end of an mRNA, for example, the 3’ end (downstream) of the 3’-UTR.

[0103] As used herein, the term “5’-cap structure”: The 5’-cap structure is typically located at the 5’ end of a mature mRNA. In some embodiments, the 5’-cap structure is linked to the 5’-end of the mRNA by a 5’-5’-triphosphate linkage. The 5’-cap structure is typically formed from a modified (e.g., methylated) ribonucleotide, especially a guanine nucleotide derivative. For example, m7GpppN (cap 0 or “cap0”, is a cap structure formed by the 5’,5’-phosphodiester linkage between the 5’-phosphate group of the hnRNA and the 5’-phosphate group of m7GTP under the action of guanylyltransferase), where N is the terminal 5’ nucleotide of the nucleic acid carrying the 5’-cap structure. In some embodiments, the 5’-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylation of the sugar 2’-OH of the first nucleotide of the hnRNA on the basis of cap 0, or “cap1”), cap 2 (a cap structure formed by further methylation of the sugar 2’-OH of the second nucleotide of the hnRNA on the basis of cap 1, or “cap2”), cap 4, cap 0 analog, cap 1 analog, cap 2 analog, or cap 4 analog.

[0104] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of a transcript and / or a polypeptide. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, in particular mRNA, in a system free of cells, e.g. in an appropriate cell extract (see, e.g. Pardi N., Muramatsu H., Weissman D., Kariko K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used for the production of a transcript is also referred to as "transcription vector", wherein regulatory sequences required for transcription are comprised. The term "transcription" encompasses "in vitro transcription".

[0105] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, express a polynucleotide or a vector. The term "host cell" includes prokaryotic cells (e.g. E. coli) or eukaryotic cells (e.g. yeast cells and insect cells). For example, cells from humans, mice, hamsters, pigs, goats, primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells and embryonic stem cells. In further embodiments, the host cell is an antigen presenting cell, in particular a dendritic cell, a monocyte or a macrophage. The nucleic acid can be present in the host cell in a single copy or in several copies. In some embodiments, the host cell can be a cell in which a polypeptide of the present application is expressed.

[0106] In the context of the present application, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. In particular, the term "plasmid" also encompasses molecules obtained by linearizing a circular plasmid, e.g. by digesting the circular plasmid with a restriction enzyme, thereby transforming the circular plasmid molecule into a linear molecule. Plasmids can replicate, i.e. be amplified in a cell independently of the genetic information stored as chromosomal DNA, and can be used for cloning, i.e. for amplifying genetic information in bacterial cells. In one alternatively specific example, the DNA plasmid is a medium copy or high copy plasmid. In another alternatively specific example the DNA plasmid is a high copy plasmid. Examples of such high copy plasmids include, e.g., pUC and pTZ plasmids or any other plasmid comprising an origin of replication supporting high copy plasmids (e.g. pMB1, pCoIE1).

[0107] The term "vaccine" is typically understood to provide at least one antigen or prophylactic or therapeutic material having the function of an antigen, which can stimulate the adaptive immune system of the body to provide an adaptive immune response.

[0108] The terms "treatment" and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The treatment of the present application can thus relate to the treatment of the state of a disease, but also to a prophylactic treatment in the sense of a complete or partial prevention of a disease or its symptoms. In some embodiments, the term "treatment" is to be understood as being therapeutic in the sense of a partial or complete cure of a disease and / or adverse effects and / or symptoms attributed to the disease. Treatment can also be prophylactic or preventative treatment, i.e. measures taken to prevent a disease, for example to prevent the onset of an infection and / or disease.

[0109] As used herein, the terms "subject," "subject," and "patient" can be used interchangeably. In certain embodiments, the subject is a mammal, such as a human, a non-human primate (e.g., simian, chimpanzee, monkey, and orangutan), a domesticated animal (including dogs and cats as well as livestock animals (e.g., horses, cows, pigs, sheep, and goats)), or another mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, and the like. In particular embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having an infectious or neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious or neoplastic disease.

[0110] As used herein, the term "administering" refers to providing or giving a subject a pharmaceutical agent by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intra-biliary duct, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom. When used to prevent a disease, disorder, condition, or symptom, administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.

[0111] Herein, some elements of the present application will be described. These elements and specific embodiments are listed together, however it is understood that they can be combined in any way and in any number to create additional embodiments. The different described examples and preferred embodiments should not be construed as limiting the present application to only the explicitly described embodiments. This specification is understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, any permutation and combination of the described elements in the present application should be considered as being disclosed by the specification of the present application. For example, in one embodiment, the nucleic acid comprises a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof, the nucleic acid is mRNA, and in another embodiment, the HSV glycoprotein or the immunogenic fragment thereof also has a mutation, then the following scenario is also an embodiment claimed by the present application: the nucleic acid comprises a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof, the HSV glycoprotein or the immunogenic fragment thereof has a mutation, the nucleic acid is mRNA.

[0112] II. Nucleic acid

[0113] The present disclosure provides a nucleic acid, the nucleic acid is a non-natural nucleic acid, the nucleic acid comprises a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof, the HSV glycoprotein is selected from one or more of the following: HSV glycoprotein D (gD), HSV glycoprotein C (gC), and HSV glycoprotein E (gE).

[0114] Herein, "HSV glycoprotein or an immunogenic fragment thereof" is a short name for HSV glycoprotein or a HSV glycoprotein immunogenic fragment. For example, HSV glycoprotein E or an immunogenic fragment thereof refers to HSV glycoprotein E or a HSV glycoprotein E immunogenic fragment.

[0115] In some embodiments, "immunogenic fragment" refers to a portion of a protein that is immunogenic and elicits a protective immune response when administered to a subject.

[0116] In some embodiments, the HSV glycoprotein is a HSV-1 glycoprotein and / or a HSV-2 glycoprotein.

[0117] In some embodiments, the above-mentioned nucleic acid comprises a polynucleotide encoding a HSV-2 glycoprotein D or an immunogenic fragment thereof.

[0118] In some embodiments, the HSV-2 glycoprotein D is a HSV-2 full-length glycoprotein D.

[0119] In some embodiments, the amino acid sequence of the HSV-2 full-length glycoprotein D is set forth in SEQ ID NO: 46.

[0120] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment comprises or is a truncated HSV-2 glycoprotein D that retains amino acid positions 26-393, 26-363, or 26-331 of HSV-2 glycoprotein D. It is noted that the amino acid position numbers 26-393, 26-363, and 26-331 herein are with reference to the corresponding amino acid positions of the HSV-2 full-length glycoprotein D set forth in SEQ ID NO: 46. Those skilled in the art will appreciate that different HSV-2 gD can have different numbering systems (e.g., additional amino acid residues are added or deleted compared to SEQ ID NO: 46). Thus, it should be understood that when a particular amino acid residue is referred to using a number, this refers not only to the amino acid residue that is precisely at that numbered position when counting from the beginning of a given amino acid sequence, but also includes any and all equivalent / corresponding amino acid residues in the amino acid sequence of any and all HSV-2 gD, even if the amino acid residue is not at the same precise numbered position (e.g., if the amino acid sequence of the HSV-2 gD is shorter or longer than SEQ ID NO: 46, or has insertions or deletions compared to SEQ ID NO: 46). In addition, the same reasoning applies to the amino acid position numbering of HSV-2 gC and HSV-2 gE below.

[0121] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment comprises or is a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95% and less than 100% (e.g., at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% and less than 100%) identical to the amino acid sequence set forth in SEQ ID NO: 1, 4, or 7. It is noted that “at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% and less than 100%” is a shorthand for at least 95% and less than 100%, at least 95.5% and less than 100%, at least 96% and less than 100%, at least 96.5% and less than 100%, at least 97% and less than 100%, at least 97.5% and less than 100%, at least 98% and less than 100%, at least 98.5% and less than 100%, at least 99% and less than 100%, or at least 99.5% and less than 100%, and the same reasoning applies elsewhere herein.

[0122] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein D comprises or is a truncated HSV-2 glycoprotein D having an amino acid sequence that is 96.8% and less than 100% (e.g., at least 96.8%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% and less than 100%) identical to the amino acid sequence set forth in SEQ ID NO: 1 or 7.

[0123] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein D comprises or is a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 96.5% and less than 100% (e.g., at least 96.5%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100%) identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0124] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein D comprises or is a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 1, 4, or 7.

[0125] In some embodiments, the HSV-2 glycoprotein or immunogenic fragment thereof further has a mutation.

[0126] In some embodiments, the HSV-2 glycoprotein D or immunogenic fragment thereof further has a mutation.

[0127] In some embodiments, the HSV-2 glycoprotein or immunogenic fragment thereof is further linked to a heterologous signal peptide.

[0128] In some embodiments, the heterologous signal peptide is located at the N-terminus or C-terminus of the HSV-2 glycoprotein or immunogenic fragment thereof.

[0129] In some embodiments, the heterologous signal peptide is located at the N-terminus of the HSV-2 glycoprotein or immunogenic fragment thereof.

[0130] In some embodiments, the HSV-2 glycoprotein D or immunogenic fragment thereof is further linked to a heterologous signal peptide.

[0131] In some embodiments, the heterologous signal peptide is located at the N-terminus or C-terminus of the HSV-2 glycoprotein D or immunogenic fragment thereof.

[0132] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein D is further linked to a heterologous signal peptide, which is located at the N-terminus of the immunogenic fragment of HSV-2 glycoprotein D.

[0133] In some embodiments, the HSV-2 glycoprotein D immunogenic fragment comprising HSV-2 glycoprotein D from position 26 to 393, from position 26 to 363, or from position 26 to 331 of HSV-2 glycoprotein D is further linked to a heterologous signal peptide. For example, the immunogenic fragment of HSV-2 glycoprotein D from position 26 to 393, from position 26 to 363, or from position 26 to 331 of HSV-2 glycoprotein D is further linked to a heterologous signal peptide only.

[0134] In some embodiments, the HSV glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 1, 4, or 7 is further linked to a heterologous signal peptide.

[0135] In some embodiments, the nucleic acid described above comprises a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof.

[0136] In some embodiments, the HSV-2 glycoprotein C is a HSV-2 full-length glycoprotein C.

[0137] In some embodiments, the HSV-2 full-length glycoprotein C has an amino acid sequence as set forth in SEQ ID NO: 47.

[0138] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment comprises or is a truncated HSV-2 glycoprotein C retaining HSV-2 glycoprotein C from position 28 to 480, from position 28 to 468, or from position 27 to 426. It is noted that the amino acid position numbers in position 28 to 480, position 28 to 468, and position 27 to 426 are referenced to the corresponding amino acid positions of the HSV-2 full-length glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 47.

[0139] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment comprises or is a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95% and less than 100% (e.g., at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100%) identical to the amino acid sequence as set forth in SEQ ID NO: 2, 5, or 8.

[0140] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment comprises or is a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 2 or 5.

[0141] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment comprises or is a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 98%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0142] In some embodiments, the HSV-2 glycoprotein C or immunogenic fragment thereof further has a mutation.

[0143] In some embodiments, the HSV-2 glycoprotein C or immunogenic fragment thereof is further linked to a heterologous signal peptide.

[0144] In some embodiments, the heterologous signal peptide is located at the N-terminus or C-terminus of the HSV-2 glycoprotein C or immunogenic fragment thereof.

[0145] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment is further linked to a heterologous signal peptide, which is located at the N-terminus of the HSV-2 glycoprotein C immunogenic fragment.

[0146] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment comprising positions 28-480, 28-468, or 27-426 of HSV-2 glycoprotein C is further linked to a heterologous signal peptide. For example, the HSV-2 glycoprotein C immunogenic fragment comprising positions 28-480, 28-468, or 27-426 of HSV-2 glycoprotein C is further linked to a heterologous signal peptide.

[0147] In some embodiments, the HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence set forth in SEQ ID NO: 2, 5, or 8 is further linked to a heterologous signal peptide.

[0148] In some embodiments, the nucleic acid described above comprises a polynucleotide encoding the HSV-2 glycoprotein E or immunogenic fragment thereof.

[0149] In some embodiments, the HSV-2 glycoprotein E is a full-length HSV-2 glycoprotein E.

[0150] In some embodiments, the amino acid sequence of the HSV-2 full-length glycoprotein E is set forth in SEQ ID NO: 48 or 49.

[0151] In some embodiments, the HSV-2 glycoprotein E immunogenic fragment comprises or is a truncated HSV-2 glycoprotein E retaining positions 21-545, 24-548, 24-442, or 24-405 of HSV-2 glycoprotein E. It is noted that the amino acid position numbering in positions 21-545 is with reference to the HSV-2 full-length glycoprotein E set forth in SEQ ID NO: 49. The amino acid position numbering in positions 24-548, 24-442, and 24-405 is with reference to the corresponding amino acid positions of the HSV-2 full-length glycoprotein E set forth in SEQ ID NO: 48.

[0152] In some embodiments, the HSV-2 glycoprotein E immunogenic fragment comprises or is a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95% and less than 100% (e.g., at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.6%, 96.8%, 97%, 97.2%, 97.5%, 98%, 98.5%, 99%, or 99.5% and less than 100%) identical to the amino acid sequence set forth in SEQ ID NO: 3, 6, 9, or 42.

[0153] In some embodiments, the HSV-2 glycoprotein E immunogenic fragment comprises or is a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 96.6%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0154] In some embodiments, the HSV-2 glycoprotein E immunogenic fragment comprises or is a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 9.

[0155] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein E comprises or is a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence set forth as SEQ ID NO: 42.

[0156] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein E comprises or is a truncated HSV-2 glycoprotein E having an amino acid sequence set forth as SEQ ID NO: 3, 6, 9, or 42.

[0157] In some embodiments, the HSV-2 glycoprotein E or immunogenic fragment thereof further has a mutation.

[0158] In some embodiments, the HSV-2 glycoprotein E or immunogenic fragment thereof is further linked to a heterologous signal peptide.

[0159] In some embodiments, the HSV-2 glycoprotein E or immunogenic fragment thereof is linked to a heterologous signal peptide, which is located at the N-terminus or C-terminus of the HSV-2 glycoprotein E or immunogenic fragment thereof.

[0160] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein E is linked to a heterologous signal peptide; the heterologous signal peptide is located at the N-terminus of the immunogenic fragment of HSV-2 glycoprotein E.

[0161] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein E comprising HSV-2 glycoprotein E positions 21-545, 24-548, 24-442, or 24-405 of HSV-2 glycoprotein E is further linked to a heterologous signal peptide. For example, the immunogenic fragment of HSV-2 glycoprotein E comprising HSV-2 glycoprotein E positions 21-545, 24-548, 24-442, or 24-405 of HSV-2 glycoprotein E is further linked to a heterologous signal peptide.

[0162] In some embodiments, the immunogenic fragment of HSV-2 glycoprotein E having an amino acid sequence set forth as SEQ ID NO: 3, 6, 9, or 42 is further linked to a heterologous signal peptide.

[0163] A "heterologous signal peptide" herein means that the signal peptide to which the HSV-2 glycoprotein or immunogenic fragment thereof is further linked is heterologous, e.g., the signal peptide to which the HSV-2 glycoprotein or immunogenic fragment thereof is further linked does not naturally (in the native state) exist in this combination, they are typically recombinant. For example, the signal peptide linked to the HSV-2 glycoprotein or immunogenic fragment thereof is a signal peptide derived from another protein (e.g., IgG) other than the HSV-2 glycoprotein. For another example, the signal peptide linked to the HSV-2 glycoprotein or immunogenic fragment thereof is a signal peptide derived from a different protein of the HSV-2 glycoprotein (e.g., a signal peptide derived from HSV-2 glycoprotein D linked to HSV-2 glycoprotein C or immunogenic fragment thereof).

[0164] In some embodiments, the heterologous signal peptide of some embodiments described above is selected from one or more of the following: an IgE signal peptide and an IgG kappa signal peptide. In some embodiments, the heterologous signal peptide of some embodiments described above is an IgE HC (Ig heavy chain epsilon-1) signal peptide. In some embodiments, the amino acid sequence of the heterologous signal peptide of some embodiments described above is MDWTWILFLVAAATRVHS. In some embodiments, the amino acid sequence of the heterologous signal peptide of some embodiments described above is METPAQLLFLLLLWLPDTTG. In some embodiments, the heterologous signal peptide of some embodiments described above is selected from one or more of the following: Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS), VSV g protein signal sequence (MKCLLYLAFLFIGVNCA), and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA).

[0165] In some embodiments, the heterologous signal peptide of some embodiments described above is an IgG kappa signal peptide.

[0166] In some embodiments, the IgG kappa signal peptide is a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31 and has the function of a signal peptide.

[0167] In some embodiments, the amino acid sequence of the IgG kappa signal peptide is set forth in SEQ ID NO: 31.

[0168] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 33, 36, or 39. It is noted that "at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical" is a shorthand for at least 95% identical, at least 95.2% identical, at least 95.5% identical, at least 95.8% identical, at least 96% identical, at least 96.2% identical, at least 96.5% identical, at least 96.8% identical, at least 97% identical, at least 97.2% identical, at least 97.5% identical, at least 97.8% identical, at least 98% identical, at least 98.2% identical, at least 98.5% identical, at least 98.8% identical, at least 99% identical, at least 99.2% identical, at least 99.5% identical, or at least 99.8% identical, and the like elsewhere herein.

[0169] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide is at least 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 33 or 39.

[0170] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide is at least 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 36.

[0171] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide is set forth as SEQ ID NO: 33, 36, or 39.

[0172] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 34, 37, or 40.

[0173] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide is at least 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 34 or 37.

[0174] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide is at least 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 40.

[0175] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide is set forth as SEQ ID NO: 34, 37, or 40.

[0176] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.6%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 35, 38, 41, or 44.

[0177] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide is at least 96.6%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identical to the amino acid sequence set forth as SEQ ID NO: 35.

[0178] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked with a heterologous signal peptide has at least 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identity to the amino acid sequence set forth as SEQ ID NO: 38 or 41.

[0179] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked with a heterologous signal peptide has at least 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% identity to the amino acid sequence set forth as SEQ ID NO: 44.

[0180] In some embodiments, the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked with a heterologous signal peptide is set forth as SEQ ID NO: 35, 38, 41, or 44.

[0181] In some embodiments, the nucleic acid described above is a polynucleotide encoding an HSV glycoprotein or an immunogenic fragment thereof.

[0182] In some embodiments, the nucleic acid described above further comprises a polynucleotide encoding a protein or polypeptide other than an HSV glycoprotein or an immunogenic fragment thereof. It is understood that the protein or polypeptide other than an HSV glycoprotein or an immunogenic fragment thereof refers to a protein or polypeptide having biological significance (e.g., immunogenicity). For example, it can be another protein of HSV (e.g., a tegument protein), a fragment thereof, a variant thereof, or a variant of a fragment thereof, another HSV glycoprotein of HSV (e.g., one or more of gI, gB, gH, gK, gL, gN, and gM of HSV), a fragment thereof, a variant thereof, or a variant of a fragment thereof. It is noted that “A, a fragment thereof, a variant thereof, or a variant of a fragment thereof” is a shorthand for A, a fragment of A, a variant of A, or a variant of a fragment of A.

[0183] In some embodiments, the nucleic acid containing a polynucleotide encoding an HSV glycoprotein or immunogenic fragment thereof is codon-optimized. Methods of codon optimization are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to: match codon frequencies of the target and host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeated codons or base extensions that can impair gene architecture or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein (e.g., glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; modulate translation rate so that various domains of the protein can fold properly; or reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, non-limiting examples including services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or patented methods.

[0184] In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0185] In some embodiments, the codon-optimized nucleic acid has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the nucleic acid prior to codon optimization.

[0186] In some embodiments, the polynucleotide encoding the heterologous signal peptide is codon-optimized.

[0187] In some embodiments, the polynucleotide encoding the heterologous signal peptide has a nucleotide sequence as set forth in SEQ ID NO: 32.

[0188] In some embodiments, the nucleic acid described above comprises a polynucleotide encoding an HSV glycoprotein or immunogenic fragment thereof, the HSV glycoprotein comprising HSV glycoprotein D, HSV glycoprotein C, and HSV glycoprotein E. That is, the nucleic acid described above comprises a polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof, a polynucleotide encoding HSV glycoprotein C or an immunogenic fragment thereof, and a polynucleotide encoding HSV glycoprotein E or an immunogenic fragment thereof, and the polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof, the polynucleotide encoding HSV glycoprotein C or an immunogenic fragment thereof, and the polynucleotide encoding HSV glycoprotein E or an immunogenic fragment thereof are on the same nucleic acid strand.

[0189] It is understood that in the above nucleic acids, whether the polynucleotide encodes an HSV glycoprotein or immunogenic fragment thereof or a polynucleotide that encodes a protein or polypeptide other than an HSV glycoprotein or immunogenic fragment thereof, there can be multiple (e.g., two or three) repeats of the unit on the same strand. For example, for a polynucleotide that encodes HSV glycoprotein D, the polynucleotide that encodes HSV glycoprotein D is referred to as "A", there can be multiple A's in the same nucleic acid. For example, for a polynucleotide that encodes HSV glycoprotein D, HSV glycoprotein C, and HSV glycoprotein E, the polynucleotide that encodes HSV glycoprotein D is referred to as "A", the polynucleotide that encodes HSV glycoprotein C is referred to as "B", and the polynucleotide that encodes HSV glycoprotein E is referred to as "C", there can be multiple A's, multiple B's, and multiple C's in the same nucleic acid, there can be one A, multiple B's, and multiple C's in the same nucleic acid, there can be one A, one B, and multiple C's in the same nucleic acid, there can be multiple A's, multiple B's, and one C in the same nucleic acid, and there can be multiple A's, one B, and one C in the same nucleic acid. For example, for a polynucleotide that encodes HSV glycoprotein D and another HSV glycoprotein of HSV (e.g., HSV gl), the polynucleotide that encodes HSV glycoprotein D is referred to as "A" and the polynucleotide that encodes the other HSV glycoprotein of HSV is referred to as "D", there can be multiple A's and multiple D's in the same nucleic acid, there can be one A and multiple D's in the same nucleic acid, and there can be multiple A's and one D in the same nucleic acid.

[0190] In some embodiments, the above nucleic acid is a nucleic acid that encodes an HSV-2 glycoprotein or immunogenic fragment thereof linked to a heterologous signal peptide.

[0191] In some embodiments, the above nucleic acid is an isolated nucleic acid.

[0192] In some embodiments, the above nucleic acid is an RNA.

[0193] In some embodiments, the nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide set forth in at least one of nucleotide sequences such as SEQ ID NOs: 10-27, 43, and 45 and encoding a corresponding HSV-2 glycoprotein immunogenic fragment of an amino acid sequence such as set forth in SEQ ID NOs: 1-9, 33-42, and 44. For example, the nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide set forth in SEQ ID NO: 10 and encoding an immunogenic fragment of HSV-2 glycoprotein D of an amino acid sequence such as set forth in SEQ ID NO: 1.

[0194] In some embodiments, the nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide set forth in SEQ ID NO: 16 and encoding a corresponding HSV-2 glycoprotein D immunogenic fragment of an amino acid sequence such as set forth in SEQ ID NO: 7.

[0195] In some embodiments, the nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide set forth in SEQ ID NO: 17 and encoding a corresponding HSV-2 glycoprotein C immunogenic fragment of an amino acid sequence such as set forth in SEQ ID NO: 8.

[0196] In some embodiments, the nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide set forth in SEQ ID NO: 18 and encoding a corresponding HSV-2 glycoprotein E immunogenic fragment of an amino acid sequence such as set forth in SEQ ID NO: 9.

[0197] In some embodiments, the above-mentioned nucleic acid comprises or is an RNA corresponding to a polynucleotide as set forth in any one of SEQ ID NOs: 10-27, 43, and 45.

[0198] In some embodiments, the RNA is an mRNA.

[0199] In some embodiments, the mRNA is a non-self-replicating mRNA.

[0200] In some embodiments, the above-mentioned nucleic acid is an RNA, which contains an open reading frame (ORF).

[0201] In some embodiments, the above-mentioned nucleic acid is an RNA, which is an mRNA.

[0202] In some embodiments, the above-mentioned nucleic acid is an mRNA, which comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail. In an alternatively specific example, the above-mentioned nucleic acid is an mRNA, which comprises a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0203] In some embodiments, the 5'-cap structure is selected from at least one of m 7 GpppG, m2 7,3′-O GpppG, m 7 Gppp(5')N1, and m 7 Gppp(m 2′-O )N1; wherein "m 7 G" represents a 7-methylguanosine cap nucleoside, "ppp" represents a triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1is the 5' most nucleotide, "G" represents a guanine nucleoside, "7" represents a methyl group at the 7-position of the guanine, and "m 2′-O " represents a methyl group at the 2'-O position of the nucleotide. In some embodiments, the 5'-cap structure is m 7 Gppp(5')N1, or m 7 Gppp(m 2′-O )N1. It is understood that in other embodiments, the 5'-cap structure is not limited to the above.

[0204] In some embodiments, the 5'-UTR corresponds to a DNA sequence as set forth in SEQ ID NO: 28.

[0205] In some embodiments, the 3'-UTR corresponds to a DNA sequence as set forth in SEQ ID NO: 29.

[0206] It should be noted that the "5'-UTR corresponding DNA sequence" refers to the nucleotide sequence of the 5'-UTR in DNA form, and the "3'-UTR corresponding DNA sequence" and the "poly(A) tail corresponding DNA sequence" below are the same. It can be understood that in other embodiments, the 5'-UTR and the 3'-UTR are not limited to the above, but can also be other, for example, the 5'-UTR and the 3'-UTR described in patents such as CN108291230A, CN104321432A, CN107849574A, etc.

[0207] In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively. In some embodiments, the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides. In one alternatively specific example, the poly(A) tail corresponding DNA sequence is shown as SEQ ID NO: 30. It can be understood that in other embodiments, the poly(A) tail is not limited to the above, but can also be other poly(A) tails, for example, the poly(A) tail described in patents such as US20170166905A1, WO2020074642A1, etc.

[0208] In some embodiments, the above-mentioned nucleic acid does not contain modified nucleotides.

[0209] In some embodiments, the above-mentioned nucleic acid contains modified nucleotides.

[0210] In some embodiments, the above-mentioned nucleic acid contains all modified nucleotides.

[0211] In some embodiments, the above-mentioned nucleic acid contains modified nucleotides. In some embodiments, the modified nucleotides include at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0212] In some embodiments, the modified nucleosides in the above-described nucleic acids are modified uridines. In some embodiments, between 0.1% and 100% of the uridines in the above-described nucleic acids are modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the uridines in the above-described nucleic acids are modified. In some embodiments, between 80% and 100% of the uridines are modified. In some embodiments, 100% of the uridines are modified. Exemplary modified uridines are selected from, but not limited to, one or more of: pseudouridine (ψ), N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxylacetic acid (cmo5U), uridine-5-oxylacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U,i.e., having the nucleobase deoxythymine), 1 -methyl-pseudouridine (m1y), 5-methyl-2-thio-uridine (m5s2U), 1 -methyl-4-thio-pseudouridine (m1s4y), 4-thio-1 -methyl-pseudouridine, 3-methyl-pseudouridine (m3y), 2-thio-1 -methyl-pseudouridine, 1 -methyl-1 -deaza-pseudouridine, 2-thio-1 -methyl-1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydrouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (y), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbomoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0213] In some embodiments, the modified nucleosides in the above-described nucleic acids are modified cytidines. In some embodiments, between 0.1% and 100% of the cytidines in the above-described nucleic acids are modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the cytidines in the above-described nucleic acids are modified. In some embodiments, between 80% and 100% of the cytidines are modified. In some embodiments, 100% of the cytidines are modified. Exemplary modified cytidines are selected from, but not limited to, one or more of: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabinocytidine, 2'-F-cytidine, and 2'-OH-arabinocytidine.

[0214] In some embodiments, the modified nucleosides in the above-described nucleic acids are modified adenosines. In some embodiments, between 0.1% and 100% of the adenosines in the above-described nucleic acids are modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the adenosines in the above-described nucleic acids are modified. In some embodiments, between 80% and 100% of the adenosines are modified. In some embodiments, between 80% and 100% of the adenosines are modified. In some embodiments, 100% of the adenosines are modified.Exemplary modified adenosines are selected from, but not limited to, one or more of: 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (mlAm), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinoadenosine, 2'-F-adenosine, 2'-OH-arabinoadenosine, and N6-(19-amino-pentadeca-oxa-nonadecyl)-adenosine.

[0215] In some embodiments, the modified nucleosides in the above-described nucleic acids are modified guanosines. In some embodiments, 0.1-100% of the guanosines in the above-described nucleic acids are modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the guanosines in the above-described nucleic acids are modified. In some embodiments, 80-100% of the guanosines are modified. In some embodiments, 80-100% of the guanosines are modified. In some embodiments, 100% of the guanosines are modified.Exemplary modified guanosines are selected from, but not limited to, one or more of: inosine (I), 1-methyl-inosine (ml), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), iso-wyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQl),

[0216] In some embodiments, the modified nucleotides in the above-described nucleic acids comprise nucleotides containing an isotope.

[0217] In some embodiments, the above-described nucleic acids comprise nucleotides containing an isotope of hydrogen. The isotope of hydrogen is not limited to deuterium, tritium. In addition, in some embodiments, the above-described nucleic acids further comprise or are nucleotides containing an isotope of other elements in addition to hydrogen, wherein the other elements include, but are not limited to, carbon, oxygen, nitrogen, and phosphorus.

[0218] In some embodiments, the above nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 10-27, 43, and 45, and encoding a corresponding HSV-2 glycoprotein immunogenic fragment of an amino acid sequence set forth in any one of SEQ ID NOs: 1-9, 33-42, and 44, and wherein all uridines in the nucleic acid are replaced with Nl-methyl pseudouridines.

[0219] In some embodiments, the above nucleic acid comprises or is an RNA corresponding to a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 10-27, 43, and 45, and encoding a corresponding HSV-2 glycoprotein immunogenic fragment of an amino acid sequence set forth in any one of SEQ ID NOs: 1-9, 33-42, and 44, and wherein all uridines in the nucleic acid are replaced with Nl-methyl pseudouridines.

[0220] In some embodiments, the above nucleic acid is an RNA, and the above nucleic acid has a length of at least 1000 nt, 1200 nt, 1500 nt, 1550 nt, 1600 nt, 1650 nt, 1700 nt, 1750 nt, 1800 nt, 1850 nt, 1900 nt, 1950 nt, 2000 nt, 2050 nt, 2100 nt, 2150 nt, 2200 nt, 2250 nt, 2300 nt, 2350 nt, 2400 nt, 2450 nt, 2500 nt, 2550 nt, 3000 nt, 3100 nt, 3200 nt, 3300 nt, 3400 nt, 3500 nt, 3600 nt, 3700 nt, 3800 nt, 3900 nt, 4000 nt, 4100 nt, 4200 nt, 4300 nt, 4400 nt, 4500 nt, 4600 nt, 4700 nt, 4800 nt, 4900 nt, 5000 nt, 5100 nt, 5200 nt, 5300 nt, 5400 nt, or 5500 nt.

[0221] In some embodiments, the above nucleic acid is an RNA, and the above nucleic acid has a length of 1000 nt to 8000 nt, 1000 nt to 6000 nt, 1200 nt to 6500 nt, 1200 nt to 6000 nt, 1200 nt to 5500 nt, 1200 nt to 5000 nt, 1200 nt to 4500 nt, 1200 nt to 4000 nt, 1200 nt to 3500 nt, 1200 nt to 3000 nt, 1200 nt to 2500 nt, 1200 nt to 2400 nt, or 1200 nt to 2000 nt.

[0222] In some embodiments, the above nucleic acid is an mRNA in Table 1. The mRNA in Table 1 is an mRNA encoding a HSV-2 glycoprotein immunogenic fragment linked with a heterologous signal peptide, which comprises a Cap1 type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO: 28, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO: 29, a poly(A) tail corresponding to the sequence set forth in SEQ ID NO: 30, a protein coding region of the immunogenic fragment of the HSV-2 glycoprotein corresponding to any one of the sequences set forth in SEQ ID NOs: 1-9 and 42, and a polynucleotide encoding an IgGk signal peptide corresponding to the sequence set forth in SEQ ID NO: 32 located at the 5' end of the protein coding region of the immunogenic fragment of the HSV-2 glycoprotein, wherein all uridines in the mRNA in Table 1 are N1-methyl pseudouridines. For example, the mRNA set forth in No. 001 comprises, in order from 5' to 3', a Cap1 type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO: 28, a protein coding region of the mRNA corresponding to the sequence set forth in SEQ ID NO: 19, which comprises a protein coding region of the HSV-2 glycoprotein D immunogenic fragment corresponding to the sequence set forth in SEQ ID NO: 10 and a polynucleotide encoding an IgGk signal peptide corresponding to the sequence set forth in SEQ ID NO: 32, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO: 29, and a poly(A) tail corresponding to the sequence set forth in SEQ ID NO: 30, and all uridines in the mRNA set forth in No. 001 are N1-methyl pseudouridines.

[0223] Table 1

[0224] In other embodiments, the above nucleic acid is DNA.

[0225] In some embodiments, the above nucleic acid is DNA, which is capable of being transcribed into RNA.

[0226] In some embodiments, the above nucleic acid is DNA, which contains an ORF.

[0227] In some embodiments, the above nucleic acid comprises or is a polynucleotide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide set forth in any one of SEQ ID NOs: 10-27, 43, and 45, and encoding a corresponding HSV-2 glycoprotein immunogenic fragment set forth in an amino acid sequence of any one of SEQ ID NOs: 1-9, 33-42, and 44.

[0228] In some embodiments, the nucleic acid comprises or is a polynucleotide having at least 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide of a nucleotide sequence as set forth in SEQ ID NO: 16 and encodes a corresponding HSV-2 glycoprotein D immunogenic fragment of an amino acid sequence as set forth in SEQ ID NO: 7.

[0229] In some embodiments, the nucleic acid comprises or is a polynucleotide having at least 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide of a nucleotide sequence as set forth in SEQ ID NO: 17 and encodes a corresponding multi-HSV-2 glycoprotein C immunogenic fragment of an amino acid sequence as set forth in SEQ ID NO: 8.

[0230] In some embodiments, the nucleic acid comprises or is a polynucleotide having at least 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% identity to a polynucleotide of a nucleotide sequence as set forth in SEQ ID NO: 18 and encodes a corresponding HSV-2 glycoprotein E immunogenic fragment of an amino acid sequence as set forth in SEQ ID NO: 9.

[0231] In some embodiments, the nucleic acid comprises or is a polynucleotide of a nucleotide sequence as set forth in at least one of SEQ ID NOs: 10-27, 43, and 45.

[0232] In some embodiments, the nucleic acid does not comprise a start codon and / or a stop codon. When used, a start codon and / or a stop codon can be added.

[0233] In some embodiments, the nucleic acid comprises a stop codon. It can be appreciated that the stop codon in the nucleic acid can be replaced with other stop codons, and of course, other stop codons can be further added.

[0234] In some embodiments, the stop codon is one or more of the following: TAG, TAA, TGA, UAA, UAG, and UGA.

[0235] In some embodiments, the stop codon in the nucleic acid can be one or more, for example, 2 or 3.

[0236] The nucleic acid of the present disclosure can induce the body to produce a significant immune response, and the body has a significant prophylactic or therapeutic effect on HSV. For example, at least one or more of the following advantages: reducing the probability of weight loss and death caused by HSV virus infection, reducing the titer of HSV virus after HSV virus infection, reducing the incidence after HSV virus infection, improving the clinical symptoms after HSV virus infection, reducing the copy number of HSV virus in dorsal root ganglion, and increasing the neutralizing antibody titer of HSV virus in serum.

[0237] III. Methods of preparing genetically engineered vectors, nucleic acids, host cells, proteins, or polypeptides

[0238] The present disclosure also provides a genetically engineered vector comprising the nucleic acid of any of the above embodiments, or a polynucleotide capable of transcribing or being transcribed and post-transcriptionally processed into the nucleic acid of any of the above embodiments.

[0239] In some embodiments, the genetically engineered vector described above is a plasmid, cosmid, virus, bacteriophage, or another vector commonly used in genetic engineering. In an alternatively specific example, the genetically engineered vector described above is a plasmid. In some embodiments, the genetically engineered vector described above further comprises at least one or more of the following: an origin of replication (ORI), a marker gene or a fragment thereof, a reporter gene or a fragment thereof, and a restriction site allowing insertion of a DNA element. In an alternatively specific example, the restriction site allowing insertion of a DNA element is a multiple cloning site (MCS).

[0240] In some embodiments, the genetically engineered vector described above is an expression vector.

[0241] In some embodiments, the genetically engineered vector described above comprises a promoter, a 5'-UTR, a polynucleotide encoding a signal peptide of any of the above embodiments, a polynucleotide encoding an HSV glycoprotein or an immunogenic fragment thereof of any of the above embodiments, a 3'-UTR, and a poly(A) tail, wherein the poly(A) tail, the promoter, the 5'-UTR, the polynucleotide encoding a signal peptide, the polynucleotide encoding an HSV glycoprotein or an immunogenic fragment thereof, and the 3'-UTR are operably linked to each other.

[0242] In other embodiments, the genetically engineered vector described above is a cloning vector.

[0243] In some embodiments, the genetically engineered vector described above comprises the nucleic acid described above without a 5'-cap structure.

[0244] The present disclosure also provides a method of producing the nucleic acid of any of the above embodiments, the method comprising the step of culturing the host cell containing the nucleic acid after introducing (e.g., in the form of a plasmid) the genetically engineered vector of any of the above embodiments into the host cell (e.g., E. coli).

[0245] In addition, the present disclosure also provides another method of producing the nucleic acid of any of the above embodiments, the method comprising the step of chemically synthesizing the nucleic acid according to the nucleotide sequence of the nucleic acid of any of the above embodiments. It is understood that the specific method of chemical synthesis can be a method known in the art, such as the solid phase phosphoramidite method.

[0246] It is understood that the method of producing the nucleic acid of any of the above embodiments is not limited to the above, but can also be other methods.

[0247] In addition, the present disclosure also provides a host cell comprising the nucleic acid of any of the above embodiments or the genetically engineered vector of any of the above embodiments.

[0248] In some embodiments, the host cell is an isolated cell.

[0249] In some embodiments, the host cell is used to store and / or amplify the nucleic acid of any of the above embodiments.

[0250] In some embodiments, the host cell is a bacterial cell. Bacterial host cells include E. coli cells, which are well known to those skilled in the art.

[0251] The host cell of the present disclosure can be prepared by transforming a competent host cell with the genetically engineered vector of any of the above embodiments. A competent host cell is a cell that has the ability to take up free extracellular genetic material (e.g., DNA plasmids) independent of sequence. Various bacterial cells known to those skilled in the art are naturally capable of taking up foreign DNA from the environment, and thus can serve as bacterial host cells according to the present disclosure. In addition, it is known to those skilled in the art that competent bacterial host cells can be obtained from naturally non-competent bacterial cells using, for example, electroporation or chemicals (e.g., treatment with calcium ions and concomitant exposure to high temperature). After uptake, the foreign DNA is preferably neither degraded nor integrated into the genome of the bacterial host cell.

[0252] In addition, the present disclosure also provides a protein or polypeptide encoded by the nucleic acid of any of the above embodiments.

[0253] In some embodiments, the protein or polypeptide encoded by the nucleic acid comprises an HSV glycoprotein or an immunogenic fragment thereof.

[0254] In some embodiments, the protein or polypeptide encoded by the nucleic acid comprises an HSV glycoprotein or an immunogenic fragment thereof linked to a heterologous signal peptide.

[0255] In addition, the present disclosure also provides an HSV immunogen comprising a polypeptide or protein comprising an HSV glycoprotein or an immunogenic fragment thereof encoded by the nucleic acid of any of the above embodiments.

[0256] In some embodiments, the HSV immunogen comprises an HSV glycoprotein or an immunogenic fragment thereof linked with a heterologous signal peptide.

[0257] In addition, the present disclosure also provides a method for preparing a protein or polypeptide, comprising: transcribing a polynucleotide encoding the protein or polypeptide (e.g., a nucleic acid in the form of DNA of any of the above embodiments) into mRNA; and translating the mRNA obtained by the transcription into a polypeptide or protein.

[0258] In some embodiments, the method for preparing a protein or polypeptide described above is performed entirely in vitro or partially in vitro.

[0259] In some embodiments, the protein or polypeptide described above is an HSV glycoprotein or an immunogenic fragment thereof linked with a heterologous signal peptide.

[0260] IV. RNA, method for preparing RNA

[0261] The present disclosure also provides a method for preparing RNA, comprising a step of transcribing the genetically engineered vector of any of the above embodiments of the present disclosure.

[0262] In some embodiments, the method for preparing RNA described above is an in vitro method. In some embodiments, the method for preparing RNA described above comprises contacting the genetically engineered vector (e.g., a plasmid) of any of the above embodiments with an RNA polymerase. In some embodiments, the method for preparing RNA described above further comprises a step of linearizing the genetically engineered vector (e.g., a plasmid). In some embodiments, the supercoiling rate of the genetically engineered vector (e.g., a plasmid) is at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, etc.) before linearization. In some embodiments, the method for preparing RNA further comprises a step of purifying the linearized genetically engineered vector. In some embodiments, the method for preparing RNA described above further comprises a step of purifying the RNA.

[0263] The present disclosure also provides another method for preparing RNA, comprising a step of preparing the RNA using a chemical synthesis method according to the nucleotide sequence of the RNA or the corresponding RNA of the DNA of any of the above embodiments. It can be understood that the specific method of the chemical synthesis method can be a method known in the art, such as a solid-phase phosphoramidite method.

[0264] In some embodiments, the RNA is mRNA.

[0265] In some embodiments, the method of making any of the above-mentioned RNAs further comprises a capping step and optionally a purification step of the capped product. In some embodiments, the cap is a Cap 1 cap. The Cap 1 cap structure is as follows:

[0266] cap G 1 G 2 = m 7 G-5'-ppp-5'-Gm2'-3'-p-[m7=7-CH3; m2'=2'-O-CH3; -ppp-= -PO2H-O-PO2H-O-PO2H)-; -p-= -PO2H-].

[0267] The capping reaction is shown as follows:

[0268] pppN1(p)Nx-OH (3') → ppN1(pN)x-OH (3') + Pi

[0269] ppN1(pN)x-OH (3') + GTP → G(5')ppp(5')N1(pN)x-OH (3') + PPi

[0270] G(5')ppp(5')N1(pN)x-OH (3') + AdoMet → m7G(5')ppp(5')N1(pN)x-OH (3') + AdoHyc

[0271] m7GpppN1(pN)x-OH (3') + AdoMet → m7Gppp[m2'-O]N1(pN)x-OH (3') + AdoHyc.

[0272] In other embodiments, the method of making the above-mentioned RNAs is a partially in vivo method. The RNA is prepared by a method comprising the following steps: preparing the genetically engineered vector of any of the above-mentioned embodiments in vitro; and introducing the genetically engineered vector into (e.g., in the form of a plasmid) in vivo. In some embodiments, the genetically engineered vector is encapsulated in a delivery vehicle. In this case, the genetically engineered vector can be delivered into the body by the delivery vehicle.

[0273] In some embodiments, the method of making the RNA of any of the above-mentioned embodiments, the RNA prepared comprises modified nucleosides or modified nucleotides. Correspondingly, the starting material for making the RNA comprises one or more modified nucleosides or nucleotides. It is understood that the amount and type of modified nucleosides or nucleotides used corresponds to the RNA to be prepared.

[0274] In addition, the present disclosure provides an RNA produced by the method of producing an RNA of any of the above embodiments. In some embodiments, the RNA is an mRNA.

[0275] V. Nucleic Acid Compositions

[0276] The present disclosure also provides a nucleic acid composition comprising one or more nucleic acids of any of the above embodiments, one or more genetically engineered vectors of any of the above embodiments, or one or more RNAs of any of the above embodiments.

[0277] In some embodiments, the nucleic acid composition comprises a plurality of nucleic acids of any of the above embodiments and each of the plurality of nucleic acids is independent.

[0278] In some embodiments, the nucleic acid composition comprises one or more of the following nucleic acids: (1) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof; (2) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof; and (3) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof.

[0279] In some embodiments, the nucleic acid composition comprises a plurality of each of the following independent nucleic acids: (1) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D or an immunogenic fragment thereof; (2) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C or an immunogenic fragment thereof; and (3) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E or an immunogenic fragment thereof.

[0280] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D or an immunogenic fragment thereof according to any of the embodiments described above; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C or an immunogenic fragment thereof according to any of the embodiments described above; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E or an immunogenic fragment thereof according to any of the embodiments described above. It is noted that the "each independently" of the plurality of nucleic acids in the nucleic acid composition means that there is no connecting element connecting the plurality of nucleic acids. In this case, the nucleic acids encoding different proteins are not on the same nucleic acid strand. For example, the nucleic acid composition comprises each of the following nucleic acids independently: a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a signal peptide-containing HSV-2 glycoprotein D, a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a signal peptide-containing HSV-2 glycoprotein C, and a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a signal peptide-containing HSV-2 glycoprotein E, in which the different nucleic acids comprising a polynucleotide encoding an immunogenic fragment of a signal peptide-containing HSV-2 glycoprotein in the nucleic acid composition are not on the same nucleic acid strand and are not connected to each other.

[0281] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0282] The signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 1; and (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical to, and less than 100% identical to, the amino acid sequence as set forth in SEQ ID NO: 1;

[0283] The signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 2; and (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 2.

[0284] The signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 3; and (2) a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.6%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 3.

[0285] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0286] The nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 10, or its corresponding RNA; (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 10, and that encodes the HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 1, or its corresponding RNA;

[0287] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 11, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 11 and that encodes an HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 2, or its corresponding RNA;

[0288] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 12, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 12 and that encodes an HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 3, or its corresponding RNA.

[0289] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0290] A signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of the following: (1) a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 1; and (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and less than 100%;

[0291] The signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 2; and (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 2.

[0292] The signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 42; and (2) a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 42.

[0293] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0294] The nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 10, or its corresponding RNA; (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 10, and that encodes the HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 1, or its corresponding RNA;

[0295] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 11, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 11 and that encodes an HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 2, or its corresponding RNA;

[0296] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 43, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 43 and that encodes an HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 42, or its corresponding RNA.

[0297] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0298] A signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one of the following: (1) a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 4; and (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95%, 95.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 4 and less than 100%;

[0299] The signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 5; and (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 5.

[0300] The signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 6; and (2) a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 6.

[0301] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0302] The nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 13, or its corresponding RNA; (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 13, and that encodes the HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 4, or its corresponding RNA;

[0303] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 14, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 14 and that encodes an HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 5, or its corresponding RNA;

[0304] A nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 15, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 15 and that encodes an HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 6, or its corresponding RNA.

[0305] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0306] A signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of the following: (1) a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 7; and (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 7 and less than 100%;

[0307] The signal peptide-containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein C having an amino acid sequence as set forth in SEQ ID NO: 8; and (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 8.

[0308] The signal peptide-containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of: (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 9; and (2) a truncated HSV-2 glycoprotein E having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, or 99.5% identical, and less than 100% identical, to the amino acid sequence as set forth in SEQ ID NO: 9.

[0309] In some embodiments, the nucleic acid composition described above comprises each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0310] The nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 16, or its corresponding RNA; (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 16, and that encodes the HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 7, or its corresponding RNA; and (3) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 16, and that encodes a truncated HSV-2 glycoprotein D having an amino acid sequence as set forth in SEQ ID NO: 7, or its corresponding RNA.

[0311] A nucleic acid comprising a polynucleotide encoding a signal peptide- containing HSV-2 glycoprotein C immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 17, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 17 and that encodes an HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 8, or its corresponding RNA.

[0312] A nucleic acid comprising a polynucleotide encoding a signal peptide- containing HSV-2 glycoprotein E immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 18, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 18 and that encodes an HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 9, or its corresponding RNA.

[0313] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D immunogenic fragment; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C immunogenic fragment; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E immunogenic fragment, wherein:

[0314] A nucleic acid comprising a polynucleotide encoding a signal peptide- containing HSV-2 glycoprotein D immunogenic fragment comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 16, or its corresponding RNA; (2) a DNA having a nucleotide sequence that is at least 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 16 and that encodes an HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 7, or its corresponding RNA;

[0315] A nucleic acid comprising a polynucleotide encoding a signal peptide- containing immunogenic fragment of HSV-2 glycoprotein C comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 17, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence that is at least 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 17 and that encodes an HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 8, or a RNA corresponding thereto;

[0316] A nucleic acid comprising a polynucleotide encoding a signal peptide- containing immunogenic fragment of HSV-2 glycoprotein E comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 18, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence that is at least 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 18 and that encodes an HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 9, or a RNA corresponding thereto.

[0317] In some embodiments, the nucleic acid compositions described above comprise each independently the following nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D or an immunogenic fragment thereof of any of the embodiments described above; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C or an immunogenic fragment thereof of any of the embodiments described above; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E or an immunogenic fragment thereof of any of the embodiments described above, wherein at least one of the signal peptides linked to the HSV glycoprotein or immunogenic fragment thereof is a heterologous signal peptide.

[0318] In some embodiments, the nucleic acid compositions described above comprise a plurality of the following nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide) of any of the embodiments described above; (2) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide) of any of the embodiments described above; and (3) a nucleic acid comprising a polynucleotide encoding a signal peptide-containing HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide) of any of the embodiments described above, and each independently between the plurality of nucleic acids in the nucleic acid composition.

[0319] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide); (2) a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide); and (3) a nucleic acid comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide).

[0320] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment comprising positions 26 to 393, positions 26 to 363, or positions 26 to 331 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment comprising positions 28 to 480, positions 28 to 468, or positions 27 to 426 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment comprising positions 21 to 545, positions 24 to 548, positions 24 to 442, or positions 24 to 405 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide), respectively.

[0321] In some embodiments, the nucleic acid compositions described above comprise each of the following separate nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment retaining positions 26 to 393 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment retaining positions 28 to 480 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment retaining positions 21 to 545 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide), respectively.

[0322] In some embodiments, the nucleic acid compositions described above comprise the following each independent nucleic acid: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment that retains positions 26 to 393 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment that retains positions 28 to 480 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment that retains positions 24 to 548 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide, such as an IgG kappa signal peptide, respectively.

[0323] In some embodiments, the nucleic acid compositions described above comprise the following each independent nucleic acid: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment that retains positions 26 to 363 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment that retains positions 28 to 468 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment that retains positions 24 to 442 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide, such as an IgG kappa signal peptide, respectively.

[0324] In some embodiments, the nucleic acid compositions described above comprise the following each independent nucleic acid: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment that retains positions 26 to 331 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment that retains positions 27 to 426 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment that retains positions 24 to 405 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide, such as an IgG kappa signal peptide, respectively.

[0325] In some embodiments, the nucleic acid compositions described above comprise the following each independent nucleic acid: (1) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D immunogenic fragment that retains positions 26 to 393 of HSV-2 glycoprotein D; (2) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C immunogenic fragment that retains positions 28 to 480 of HSV-2 glycoprotein C; and (3) a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E immunogenic fragment that retains positions 24 to 548 of HSV-2 glycoprotein E, wherein each HSV-2 glycoprotein immunogenic fragment is further linked to a heterologous signal peptide, such as an IgG kappa signal peptide, respectively.

[0326] The HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 33; and (2) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 33;

[0327] The HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 34; and (2) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 34;

[0328] The HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 35; and (2) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 35.

[0329] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 33; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 34; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 35.

[0330] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0331] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 19, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 19, and that encodes a HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence of SEQ ID NO: 33, or its corresponding RNA;

[0332] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 20, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 20, and that encodes a HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence of SEQ ID NO: 34, or its corresponding RNA;

[0333] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 21, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 21, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 35, or its corresponding RNA.

[0334] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0335] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or its corresponding RNA; the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or its corresponding RNA; and the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 21, or its corresponding RNA.

[0336] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0337] The HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 33; and (2) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 33;

[0338] The HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 34; and (2) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 34.

[0339] The HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 44; and (2) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 44.

[0340] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 33; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 34; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 44.

[0341] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0342] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 19, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 19, and that encodes a HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence of SEQ ID NO: 33, or its corresponding RNA;

[0343] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 20, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 20, and that encodes a HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence of SEQ ID NO: 34, or its corresponding RNA;

[0344] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 45, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 45, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 44, or its corresponding RNA.

[0345] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0346] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or its corresponding RNA; the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or its corresponding RNA; and the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 45, or its corresponding RNA.

[0347] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0348] The HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 36; and (2) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 36;

[0349] The HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 37; and (2) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 37.

[0350] The HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 38; and (2) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 38.

[0351] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 36; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 37; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 38.

[0352] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0353] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 22, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identical to the nucleotide sequence of SEQ ID NO: 22, and that encodes a HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence of SEQ ID NO: 36, or its corresponding RNA;

[0354] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 23, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identical to the nucleotide sequence of SEQ ID NO: 23, and that encodes a HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence of SEQ ID NO: 37, or its corresponding RNA;

[0355] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 24, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 24, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 38, or its corresponding RNA.

[0356] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0357] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 22, or its corresponding RNA; the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 23, or its corresponding RNA; and the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 24, or its corresponding RNA.

[0358] In some embodiments, the nucleic acid compositions described above comprise each of the following independent nucleic acids: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0359] The HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 39; and (2) an HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 39.

[0360] The HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 40; and (2) an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 40.

[0361] The HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached is one or more of: (1) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 41; and (2) an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence that is at least 95%, 95.2%, 95.5%, 95.8%, 96%, 96.2%, 96.5%, 96.8%, 97%, 97.2%, 97.5%, 97.8%, 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, or 99.8% and less than 100% identical to the amino acid sequence as set forth in SEQ ID NO: 41.

[0362] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 39; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 40; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide having an amino acid sequence of SEQ ID NO: 41.

[0363] In some embodiments, the nucleic acid compositions described above comprise each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0364] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 25, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 25, and that encodes a HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence of SEQ ID NO: 39, or its corresponding RNA;

[0365] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence of SEQ ID NO: 26, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence of SEQ ID NO: 26, and that encodes a HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence of SEQ ID NO: 40, or its corresponding RNA;

[0366] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 27, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 27, and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 41, or a RNA corresponding thereto.

[0367] In some embodiments, the nucleic acid composition described above comprises each of the following nucleic acids independently: (1) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide; (2) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide; and (3) a nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide, wherein:

[0368] The nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 25, or a RNA corresponding thereto; the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 26, or a RNA corresponding thereto; and the nucleic acid comprising a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises a DNA having a nucleotide sequence as set forth in SEQ ID NO: 27, or a RNA corresponding thereto.

[0369] In some embodiments, the nucleic acid of the polynucleotide comprised by the nucleic acid composition described above is RNA. In an alternative specific example, the nucleic acid of the polynucleotide comprised by the nucleic acid composition described above is mRNA. In an alternative specific example, the nucleic acid of the polynucleotide comprised by the nucleic acid composition described above is non-self-replicating mRNA.

[0370] In some embodiments, the nucleic acid composition described above comprises or is each of the following mRNAs independently: (i) an mRNA comprising a polynucleotide encoding a HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide according to any of the embodiments described above; (ii) an mRNA comprising a polynucleotide encoding a HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide according to any of the embodiments described above; and (iii) an mRNA comprising a polynucleotide encoding a HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide according to any of the embodiments described above.

[0371] In some embodiments, at least one of the nucleic acids in the nucleic acid compositions described above is a non-natural nucleic acid.

[0372] In some embodiments, all of the nucleic acids in the nucleic acid compositions described above are non-natural nucleic acids.

[0373] In some embodiments, the nucleic acid compositions described above further comprise nucleic acids encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof (e.g., nucleic acids encoding other immunogens) and / or other substances (e.g., buffers or lyoprotectants, etc.).

[0374] In some embodiments, the nucleic acid compositions described above further comprise nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof, or genetically engineered vectors comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof. Other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof are described above and are not repeated here.

[0375] In some embodiments, the nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof are RNAs. In an alternative specific example, the nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof are mRNAs.

[0376] In some embodiments, the nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof comprised in the nucleic acid compositions described above are one nucleic acid, e.g., a nucleic acid comprising polynucleotides encoding one other protein or polypeptide than the HSV glycoprotein or immunogenic fragment thereof. It is understood that the nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof comprised in the nucleic acid compositions described above are not limited to one, but can be multiple. For example, the nucleic acids comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof comprised in the nucleic acid compositions described above are multiple, and the multiple nucleic acids are independent of each other. It is understood that the genetically engineered vectors comprising polynucleotides encoding other proteins or polypeptides than the HSV glycoprotein or immunogenic fragment thereof are also not limited to one, but can be multiple, and the multiple genetically engineered vectors are also independent of each other.

[0377] The nucleic acid composition described above comprises the nucleic acid described above, and has at least one of the following advantages: reducing the probability of weight loss and death caused by HSV virus infection, reducing the titer of HSV virus after HSV virus infection, reducing the morbidity after HSV virus infection, improving the clinical symptoms after HSV virus infection, reducing the copy number of HSV virus in dorsal root ganglion, and increasing the neutralizing antibody titer of HSV virus in serum.

[0378] Six, pharmaceutical composition

[0379] The present disclosure also provides a pharmaceutical composition comprising the nucleic acid of any of the embodiments described above, the nucleic acid composition of any of the embodiments described above, the genetically engineered vector of any of the embodiments described above, the protein or polypeptide encoded by the nucleic acid of any of the embodiments described above, the immunogen of any of the embodiments described above, or the RNA of any of the embodiments described above.

[0380] In some embodiments, the pharmaceutical composition described above further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government of the United States (e.g., the China Food and Drug Administration (CFDA), the U.S. Food and Drug Administration (FDA)) or listed in the public recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopeia). The term "pharmaceutically acceptable carrier" refers to a substance that can be administered with the nucleic acid, genetically engineered vector, nucleic acid composition, immunogen, RNA, or protein or polypeptide of the present disclosure, including but not limited to a delivery vehicle, a diluent, a sweetener, a flavoring agent, a wetting agent, an adjuvant, a glidant, a preservative, a dye / colorant, a surfactant, a dispersant, a suspending agent, a stabilizer, an isotonic agent, a solvent, or an emulsifying agent.

[0381] In some embodiments, the pharmaceutical composition described above comprises a pharmaceutically acceptable carrier and one or more of the nucleic acid of any of the embodiments described above, one or more of the genetically engineered vector of any of the embodiments described above, one or more of the nucleic acid composition of any of the embodiments described above, one or more of the immunogen of any of the embodiments described above, one or more of the protein or polypeptide encoded by the nucleic acid of any of the embodiments described above, or one or more of the RNA of any of the embodiments described above, and a pharmaceutically acceptable carrier.

[0382] In some embodiments, the pharmaceutical composition described above comprises a pharmaceutically acceptable carrier and one of the nucleic acid of any of the embodiments described above, one of the genetically engineered vector of any of the embodiments described above, one of the nucleic acid composition of any of the embodiments described above, one of the immunogen of any of the embodiments described above, one of the protein or polypeptide encoded by the nucleic acid of any of the embodiments described above, or one of the RNA of any of the embodiments described above, and a pharmaceutically acceptable carrier.

[0383] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a plurality of nucleic acids of any of the above embodiments, a plurality of genetically engineered vectors of any of the above embodiments, a plurality of immunogens of any of the above embodiments, a plurality of proteins or polypeptides encoded by nucleic acids of any of the above embodiments, or a plurality of RNAs of any of the above embodiments.

[0384] In some embodiments, the pharmaceutically acceptable carrier is a delivery vehicle.

[0385] In some embodiments, the pharmaceutically acceptable carrier is a delivery vehicle, and the nucleic acid of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the protein or polypeptide encoded by nucleic acids of any of the above embodiments, the immunogen of any of the above embodiments, or the RNA of any of the above embodiments is formulated in the delivery vehicle.

[0386] In some embodiments, the delivery vehicle is selected from a plurality of or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles. In some embodiments, the delivery vehicle is selected from one of the following: lipid nanoparticles, liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.

[0387] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP).

[0388] In some embodiments, a lipid nanoparticle refers to a particle having a size in the nanometer range (e.g., lnm ~ 1000 nm), which includes one or more lipids.

[0389] In some embodiments, the average diameter of the lipid nanoparticle is 20-800 nm, 20-500 nm, 20-400 nm, 20-300 nm, 20-200 nm, 20-100 nm, 30-700 nm, 30-500 nm, 30-300 nm, 30-200 nm, 30-100 nm, 40-800 nm, 40-600 nm, 40-500 nm, 40-300 nm, 40-200 nm, 40-100 nm, 50-800 nm, 50-600 nm, 50-500 nm, 50-400 nm, 50-300 nm, 50-200 nm, 50-100 nm, 60-800 nm, 60-600 nm, 60-500 nm, 60-400 nm, 60-300 nm, 60-200 nm, or 60-100 nm. In some alternative specific examples, the average diameter of the lipid nanoparticle is 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm, 231 nm, 236 nm, 241 nm, 246 nm, or 249 nm. In this context, the average diameter of the lipid nanoparticle can be expressed as the z-average determined by dynamic light scattering.

[0390] In some embodiments, the lipid nanoparticle comprises one of: a cationic lipid nanoparticle, a solid lipid nanoparticle (SLN), a nanostructured lipid carrier (NLC), a non-lamellar lipid nanoparticle. In one alternative specific example, the lipid nanoparticle is a cationic lipid nanoparticle.

[0391] In some embodiments, the lipid nanoparticle contains one or more of the following: an ionizable lipid, a helper lipid, a structural lipid, and a polymer-lipid.

[0392] In some embodiments, the lipid nanoparticle contains an ionizable lipid.

[0393] In some embodiments, the lipid nanoparticle contains an ionizable lipid, a helper lipid, a structural lipid, and a polymer-lipid.

[0394] The term "ionizable lipid" refers to a lipid that becomes positively charged when the pH is lowered below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. At a pH below the pKa, the positively charged lipid is capable of binding to a negatively charged nucleic acid. In certain embodiments, the ionizable lipid comprises a zwitterionic lipid.

[0395] In some embodiments, the ionizable lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0396] wherein:

[0397] L 3 and L 4 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, or C2-C 12 alkynylene; in some embodiments, L 3 and L 4 are each independently C3-C 10 alkylene, C3-C 10 alkenylene, or C3-C 10 alkynylene; in some embodiments, L 3 and L 4 are each independently C3-C 10 alkylene; in some embodiments, L 3 and L 4 are each independently C5-C8alkylene;

[0398] G 4 and G 5 are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, or -S-C(=O)-; in some embodiments, G 4 and G 5 are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, or -O-; in some embodiments, G4 and G 5 are the same or different, each independently selected from -0-(C=0)- or -(C=0)-0-;

[0399] R 18 and R 19 are the same or different, each independently C5-C 27 alkyl, or C5-C 27 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different, each independently C8-C 20 alkyl or C8-C 20 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different, each independently C9-C 17 alkyl or C9-C 18 alkenyl containing one or two double bonds; in some embodiments, R 18 and R 19 are the same or different, each independently

[0400] R 20 is halogen, hydroxyl, cyano, C1-C6alkyl, nitro, C 1- C6alkoxy, C1-C6alkylcarbonyloxy, C1-C6alkoxycarbonyl, C1-C6alkylaminocarbonyl, or C1-C6alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxyl, cyano, C1-C6alkoxy, C1-C6alkylcarbonyloxy, C1-C6alkoxycarbonyl, C1-C6alkylaminocarbonyl, or C1-C6alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxyl, cyano, C1-C4alkoxy, C1-C4alkylcarbonyloxy, C1-C4alkoxycarbonyl, C1-C4alkylaminocarbonyl, or C1-C4alkylcarbonylamino; in some embodiments, R 20 is fluorine, hydroxyl, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl, or acetylamino;

[0401] z is 1, 2, or 3.

[0402] In some embodiments, the ionizable lipid comprises or is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0403] In some embodiments, the ionizable lipid comprises or is the following compound, or a pharmaceutically acceptable salt thereof:

[0404] In some embodiments, the ionizable lipid is one or more of ALC-0315 (CAS No. 2036272-55-4), SM-102 (CAS No. 2089251-47-6), and compound (IV-1-1).

[0405] In some embodiments, the helper lipid of the lipid nanoparticle comprises a phospholipid. The phospholipid is typically semi-synthetic, and can also be of natural origin or chemically modified. In an alternative specific example, the helper lipid of the lipid nanoparticle is a phospholipid. In some embodiments, the phospholipid of the lipid nanoparticle comprises one or more of DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1’-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-0-4’-(N,N,N-trimethyl)homoserine), and lysophospholipid. In some embodiments, the helper lipid of the lipid nanoparticle is selected from one or more of DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and / or DOPE.

[0406] In some embodiments, the structural lipid of the lipid nanoparticle comprises a sterol. In an alternative specific example, the structural lipid of the lipid nanoparticle is a sterol. In some embodiments, the sterol of the lipid nanoparticle comprises one or more of 20a-hydroxycholesterol, cholesterol, cholesterol ester, a sterol hormone, a sterol vitamin, a bile acid, ergosterol, beta-sitosterol, and an oxidized cholesterol derivative. In some embodiments, the structural lipid of the lipid nanoparticle comprises at least one of cholesterol, cholesterol ester, a sterol hormone, a sterol vitamin, and a bile acid. In some embodiments, the structural lipid of the lipid nanoparticle is cholesterol. In an alternative specific example, the structural lipid of the lipid nanoparticle is high purity cholesterol, particularly injection grade high purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20a-hydroxycholesterol.

[0407] A polymer-lipid refers to a conjugate comprising a polymer and a lipid coupled to the polymer. A polymer-lipid (e.g., a polyethylene glycol-lipid) in a lipid nanoparticle can improve the stability of the lipid nanoparticle in vivo.

[0408] In some embodiments, the lipid of the polymer-lipid used to form the lipid nanoparticle comprises one or more of: myristoyl glycerol (1,2-dimyristoyl-sn-glycerol, DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipid, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and dipalmitoyl-rac-glycero (DPG).

[0409] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticle comprises one or both of: a hydrophilic polymer and an amphiphilic polymer.

[0410] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticle is a hydrophilic polymer. In other embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticle is an amphiphilic polymer.

[0411] In some embodiments, the hydrophilic polymer comprises one or more of: polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47.

[0412] Correspondingly, the polymer-lipid comprises one or more of: polyethylene glycol-lipid (PEG-lipid), poly(oxazolines)-lipid, poly(glycerols)-lipid, poly(hydroxypropyl methacrylate)-lipid, poly(2-hydroxyethyl methacrylate)-lipid, poly(N-(2-hydroxypropyl)methacrylamide)-lipid, poly(vinylpyrrolidone)-lipid, poly(N,N-dimethyl acrylamide)-lipid, poly(N-acryloyl morpholine)-lipid, glycosaminoglycans-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-like polypeptides-lipid, serum albumin-lipid, and CD47-lipid. It is noted that “PEG-lipid” is a conjugate of polyethylene glycol and a lipid, “poly(oxazolines)-lipid” refers to a conjugate formed by coupling poly(oxazolines) with a lipid, “poly(glycerols)-lipid” refers to a conjugate formed by coupling poly(glycerols) with a lipid, and so on for other polymer-lipids. In one alternative specific example, the hydrophilic polymer comprises polyethylene glycol.

[0413] In some embodiments, the polymer-lipid comprises a PEG-lipid. In an alternative specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid comprises one or more of: myristoyl glycerol diester-PEG (DMG-PEG), distearoyl phosphatidyl ethanolamine-PEG (DSPE-PEG), diacylglycerol-PEG (DAG-PEG), dialkyloxypropyl-PEG (DAA-PEG), phospholipid-PEG, ceramide-PEG (Cer-PEG), 1,2-distearoyl-rac-glycerol-PEG (DSG-PEG), and 1,2-dipalmitoyl-rac-glycerol-PEG (DPG-PEG). The PEG-lipid is preferably DMG-PEG, DSG-PEG, DPG PEG. DMG-PEG is a polyethylene glycol derivative of 1,2-dimyristate glycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000-5000. In an alternative specific example, the average molecular weight of the PEG in the PEG-lipid is about 2000. In some embodiments, the amphiphilic polymer comprises one or more of: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-base polymers, and phosphorylcholine polymers.In some embodiments, the amphiphilic polymer comprises one or more of: poly(carboxybetaine acrylamide) (pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylpyridine.

[0414] Correspondingly, the polymer-lipid comprises one or more of: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-based polymer-lipid, and phosphocholine-based polymer-lipid. In some embodiments, the polymer-lipid comprises one or more of: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethyl phosphorylcholine)-lipid, poly(vinyl-pyridinio propanesulfonate)-lipid, poly(carboxybetaine) based on vinylimidazole-lipid, poly(sulfobetaine) based on vinylimidazole-lipid, poly(sulfobetaine) based on vinylpyridine-lipid.

[0415] In some embodiments, the PEG-lipid is capable of mitigating or avoiding accelerated blood clearance (ABC).

[0416] In some embodiments, the PEG-lipid is a compound of Formula (V), or a salt thereof, or a stereoisomer thereof:

[0417] wherein:

[0418] R 3 is OR 0 ;

[0419] R 0 is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

[0420] g is an integer between 1 and 100;

[0421] L1is optionally substituted C1-C 10 alkylene, wherein at least one methylene of the optionally substituted C1-C 10 alkylene is independently replaced with optionally substituted carbocyclylidenyl, optionally substituted heterocyclylidenyl, optionally substituted arylidenyl, optionally substituted heteroarylidenyl, -O-, -N(R M )-, -S-, -C(O)-, -C(O)N(R M )-, -NR M C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-, -NR M C(O)O-, or -NR M C(O)N(R M )-;

[0422] D is a group obtained by click chemistry or a group cleavable under physiological conditions;

[0423] h is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0424] A is

[0425] each L2is independently a bond or optionally substituted C1-C6alkylene; optionally one methylene of the optionally substituted C1-C6alkylene is replaced with -O-, -N(R M )-, -S-, -C(O)-, -C(O)N(R M )-, -NR M C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-, -NR M C(O)O-, or -NR M C(O)N(R M )-;

[0426] each R 2 is independently optionally substituted C1-C 30 alkyl, optionally substituted C1-C 30 alkenyl, or optionally substituted C1-C 30 alkynyl; optionally one or more methylenes of R 2 are independently replaced with optionally substituted carbocyclylidenyl, optionally substituted heterocyclylidenyl, optionally substituted arylidenyl, optionally substituted heteroarylidenyl, -N(R M)-, -O-, -S-, -C(O)-, -C(O)N(R M )-, -NR M C(O)-, -NR M C(O)N(R M )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-, -NR M C(O)O-, -C(O)S-, -SC(O)-, -C(=NR M )-, -C(=NR M )N(R M )-, -NR M C(=NR M )-, -NR M C(=NR M )N(R M )-, -C(S)-, -C(S)N(R M )-, -NR M C(S)-, -NR M C(S)-, -NR M C(S)N(R M )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R M )S(O)-, S(O)N(R M )-, -N(R M )S(O)N(R M )-, -OS(O)N(R M )-, -N(R M )S(O)O-, -S(O)2-, N(R M )S(O)2-, -S(O)2N(R M )-, -N(R M )S(O)2N(R M )-, -OS(O)2N(R M )- or -N(R M )S(O)2O-;

[0427] each R M is independently hydrogen, optionally substituted alkyl or a nitrogen protecting group;

[0428] Ring B is an optionally substituted carbocyclic ring, an optionally substituted heterocyclic ring, an optionally substituted aryl group or an optionally substituted heteroaryl group; and

[0429] k is equal to 1 or 2.

[0430] In some embodiments, the compound of Formula (V) is a PEG-OH lipid (i.e., R 0 is H.

[0431] In some embodiments, the PEG-lipid is a compound represented by Formula (V-1), or a salt thereof, or a stereoisomer thereof:

[0432] wherein:

[0433] R 3 is OR 0 ;

[0434] R 0 is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

[0435] g is an integer between 1 and 100;

[0436] R 5 is optionally substituted C 10 -C 40 alkyl, optionally substituted C 10 -C 40 alkenyl, or optionally substituted C 10 -C 40 alkynyl; optionally, one or more methylene groups of R 5 are independently replaced by optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R M )-, -O-, -S-, -C(O)-, -C(O)N(R M )-, -NR M C(O)-, -NR M C(O)N(R M )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-, -NR M C(O)O-, -C(O)S-, -SC(O)-, -C(=NR M )-, -C(=NR M )N(R M )-, -NR M C(=NR M )-, -NR M C(=NR M )N(R M )-, -C(S)-, -C(S)N(R M )-, -NR M C(S)-, -NR M C(S)-, -NR M C(S)N(R M)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R M )-, -N(R M )-, -N(R M )-, -N(R M )-, -OS(O)N(R M )-, -N(R M )-, -N(R M )-, -N(R M )-, -N(R M )-, -N(R M )-, -OS(O)2N(R M )-, or -N(R M )- is replaced with -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R

[0437] each R M is independently hydrogen, alkyl, or a nitrogen protecting group.

[0438] In some embodiments, the compound of Formula (V-1) is a PEG-OH lipid (i.e., R 0 is H).

[0439] Further, in some embodiments, the polymeric lipid can also be any one or more of the PEG-lipids in Hoang Thi, Thai Thanh, et al. Polymers, 12(2), 298 (2020), which is incorporated by reference herein.

[0440] In some embodiments, the PEG-lipid is a compound of Formula (VI), or a salt thereof, or a stereoisomer thereof:

[0441] wherein R 20 is H-, HO-Z8-Q3-, or H-Z8-Q3-,

[0442] Z8is -L5-CH(OR 22 )-L6-, -L5-C(=O)-L6-, -L5-CH(R 22 )-L6-, -L5-, or a bond,

[0443] Q3is -C(=O)-, -OC(=O)-, or a bond,

[0444] L4is C 1～ C 20 alkylene, or a bond,

[0445] A 11 -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -S-, or a bond,

[0446] R 21 C 4～ C 24 hydrocarbyl,

[0447] L4and R 21 total number of carbon atoms is 10-35,

[0448] L5is C 1～ C4alkylene,

[0449] L6is C 1～ C4alkylene,

[0450] R 22 H- or C1-C3 hydrocarbyl,

[0451] n is any integer from 30-60.

[0452] In some embodiments, the PEG-lipid is a compound of formula (VI-1), or a salt thereof, or a stereoisomer thereof:

[0453] wherein L4, R 21 and n are as defined above for formula (VI).

[0454] In some embodiments, in the compound of formula (VI) or (VI-1), the total number of carbon atoms of L4and R 21 is 11-22. For example, the total number of carbon atoms of L4and R 21 is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0455] In some embodiments, the PEG-lipid is compound 3-1, a salt thereof, or an isomer thereof:

[0456] In some embodiments, the PEG-lipid is compound 3-2, a salt thereof, or an isomer thereof:

[0457] In some embodiments, the compound of formula (VI-1) is prepared by the following synthetic route:

[0458] In some embodiments, the lipid nanoparticle contains compound (IV-1-1), a phospholipid, a structural lipid, and a PEG-lipid.

[0459] In some embodiments, the lipid nanoparticle contains compound (IV-1-1), a phospholipid, cholesterol, and a PEG-lipid.

[0460] In some embodiments, the lipid nanoparticle contains compound (IV-1-1), DSPC, cholesterol, and a PEG-lipid.

[0461] In some embodiments, the lipid nanoparticle comprises an ionizable lipid, a helper lipid, a structural lipid, and a polymer-lipid, the ionizable lipid being present at 25 mol% to 75 mol% of the total lipid present in the lipid nanoparticle, the helper lipid being present at 0 mol% to 45 mol% of the total lipid present in the lipid nanoparticle, the structural lipid being present at 0 mol% to 60 mol% of the total lipid present in the lipid nanoparticle, and the polymer-lipid being present at 0.5 mol% to 5 mol% of the total lipid present in the lipid nanoparticle.

[0462] In some embodiments, the ionizable lipid in the above lipid nanoparticle is present at 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol%, or 50 mol% to 55 mol% of the total lipid present in the lipid nanoparticle.

[0463] In some embodiments, the helper lipid (e.g., DSPC) in the above lipid nanoparticle is present at 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol%, or 5 mol% to 25 mol% of the total lipid present in the lipid nanoparticle.

[0464] In some embodiments, the structural lipid (e.g., cholesterol) in the above lipid nanoparticle is present at 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 40 mol% of the total lipid present in the lipid nanoparticle.

[0465] In some embodiments, the polymer-lipid (e.g., PEG-lipid) in the above lipid nanoparticle is present at 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol%, or 1.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticle.

[0466] In some embodiments, the non-lamellar lipid nanoparticle is selected from one of the following: ethosomes and echogenic liposomes.

[0467] In some embodiments, the delivery vehicle is a liposome. The liposome encapsulates the nucleic acid, the gene engineering vector, the nucleic acid encoded protein or polypeptide, the immunogen, the RNA, or the nucleic acid composition of any of the above embodiments in a vesicle formed by a phospholipid bilayer membrane. In some embodiments, the components of the liposome include phospholipids and cholesterol.

[0468] In some embodiments, the delivery vehicle is a cationic protein. In some embodiments, the cationic protein includes, but is not limited to, protamine.

[0469] In some embodiments, the delivery vehicle is a polymer. In some embodiments, the polymer as a delivery vehicle is a lipopolyplex (LPP) and / or a hyaluronic acid polymer (e.g., a hyaluronic acid gel). In an alternative specific example, the polymer as a delivery vehicle is a lipopolyplex or a hyaluronic acid gel. The lipopolyplex is a double-layer structure with a nucleic acid (e.g., mRNA) encapsulated by a polymer as the inner core and a lipid (e.g., a phospholipid) as the outer shell.

[0470] It can be appreciated that the delivery vehicle suitable for the present disclosure is not limited to the above, but can also be other substances capable of transporting the nucleic acid of any of the above embodiments, the gene engineering vector of any of the above embodiments, the nucleic acid encoded protein or polypeptide of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, or the nucleic acid composition of any of the above embodiments into the body. For example, vesicles (e.g., exosomes) and the like.

[0471] In some embodiments, the pharmaceutical composition comprises one of the nucleic acid of any of the above embodiments, the gene engineering vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, or the RNA of any of the above embodiments formulated (e.g., encapsulated) in a delivery vehicle (e.g., LNPs).

[0472] In some embodiments, the pharmaceutical composition comprises a plurality of the nucleic acid of any of the above embodiments, a plurality of the gene engineering vector of any of the above embodiments, a plurality of the RNA of any of the above embodiments, or a plurality of the nucleic acid composition of any of the above embodiments formulated (e.g., encapsulated) in a delivery vehicle (e.g., LNPs).

[0473] In some embodiments, the above pharmaceutical composition comprises multiple nucleic acids of any of the above embodiments co-formulated within a delivery vehicle (e.g., LNPs). That is, a single delivery vehicle of the above pharmaceutical composition is formulated (e.g., encapsulated) with multiple nucleic acids.

[0474] In some embodiments, the above pharmaceutical composition comprises multiple nucleic acids of any of the above embodiments formulated individually within a delivery vehicle (e.g., LNPs). That is, a single delivery vehicle of the above pharmaceutical composition is formulated (e.g., encapsulated) with one nucleic acid.

[0475] In some embodiments, the above pharmaceutical composition comprises the following three nucleic acids co-formulated or formulated individually within a delivery vehicle (e.g., LNPs): (1) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof linked to a signal peptide (e.g., a heterologous signal peptide); (2) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein C or an immunogenic fragment thereof linked to a signal peptide (e.g., a heterologous signal peptide); and (3) a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein E or an immunogenic fragment thereof linked to a signal peptide (e.g., a heterologous signal peptide).

[0476] In some embodiments, the above pharmaceutical composition comprises the following three nucleic acids co-formulated or formulated individually within a delivery vehicle (e.g., LNPs): a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide), a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide), and a nucleic acid of any of the above embodiments comprising a polynucleotide encoding HSV glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide (e.g., an IgG kappa signal peptide).

[0477] In some embodiments, the nucleic acid formulated (e.g., encapsulated) within a delivery vehicle is RNA.

[0478] In some embodiments, the nucleic acid formulated (e.g., encapsulated) within a delivery vehicle is mRNA. In some embodiments, the mRNA is non-self-replicating mRNA.

[0479] In some embodiments, the pharmaceutical composition described above comprises three nucleic acids described below, which are co-formulated within the LNPs (i.e., the three nucleic acids are encapsulated within a single lipid nanoparticle) or separately formulated within the LNPs (i.e., one of the three mRNAs is encapsulated within a single lipid nanoparticle): (i) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide; (ii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide; and (iii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide, wherein:

[0480] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide is RNA comprising one or both of: (1) RNA corresponding to the DNA of SEQ ID NO: 19; and (2) RNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 19 and that encodes an HSV-2 glycoprotein D immunogenic fragment having the amino acid sequence of SEQ ID NO: 33;

[0481] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide is RNA comprising one or both of: (1) RNA corresponding to the DNA of SEQ ID NO: 20; and (2) RNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 20 and that encodes an HSV-2 glycoprotein C immunogenic fragment having the amino acid sequence of SEQ ID NO: 34; and

[0482] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or both of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 21; and (2) RNA corresponding to a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 21 and that encodes an amino acid sequence of an HSV-2 glycoprotein E immunogenic fragment set forth in SEQ ID NO: 35.

[0483] In some embodiments, the pharmaceutical composition described above comprises three nucleic acids described below, which are co-formulated within the LNPs (i.e., the three nucleic acids are encapsulated within a single lipid nanoparticle) or which are separately formulated within the LNPs (i.e., one of the three mRNAs is encapsulated within a single lipid nanoparticle): (i) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide; (ii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide; and (iii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide, wherein:

[0484] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 19; and (2) RNA corresponding to a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 19 and that encodes an amino acid sequence of an HSV-2 glycoprotein D immunogenic fragment set forth in SEQ ID NO: 33;

[0485] An mRNA containing a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of the following: (1) RNA corresponding to the DNA of SEQ ID NO: 20; and (2) RNA corresponding to a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 20 and that encodes an HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence of SEQ ID NO: 34.

[0486] An mRNA containing a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of the following: (1) RNA corresponding to the DNA of SEQ ID NO: 45; and (2) RNA corresponding to a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 45 and that encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence of SEQ ID NO: 44.

[0487] In some embodiments, the pharmaceutical composition described above comprises the following three nucleic acids, which are co-formulated within the LNPs (i.e., the following three nucleic acids are encapsulated within a single lipid nanoparticle) or the following three nucleic acids are separately formulated within the LNPs (i.e., a single lipid nanoparticle encapsulates one of the following three mRNAs): (i) an mRNA containing a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide; (ii) an mRNA containing a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide; and (iii) an mRNA containing a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide, wherein:

[0488] An mRNA containing a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 22; and (2) RNA corresponding to the DNA of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 22 and encoding an HSV-2 glycoprotein D immunogenic fragment of the amino acid sequence set forth in SEQ ID NO: 36;

[0489] An mRNA containing a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 23; and (2) RNA corresponding to the DNA of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 23 and encoding an HSV-2 glycoprotein C immunogenic fragment of the amino acid sequence set forth in SEQ ID NO: 37; and

[0490] An mRNA containing a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 24; and (2) RNA corresponding to the DNA of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 24 and encoding an HSV-2 glycoprotein E immunogenic fragment of the amino acid sequence set forth in SEQ ID NO: 38.

[0491] In some embodiments, the pharmaceutical composition described above comprises the following three nucleic acids, which are co-formulated within the LNPs (i.e., the following three nucleic acids are encapsulated within a single lipid nanoparticle) or the following three nucleic acids are separately formulated within the LNPs (i.e., one of the following three mRNAs is encapsulated within a single lipid nanoparticle): (i) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide; (ii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide; and (iii) an mRNA comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide, wherein:

[0492] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein D or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 25; and (2) RNA corresponding to DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 25 and that encodes an HSV-2 glycoprotein D immunogenic fragment of the amino acid sequence set forth in SEQ ID NO: 39;

[0493] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein C or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 26; and (2) RNA corresponding to DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 26 and that encodes an HSV-2 glycoprotein C immunogenic fragment of the amino acid sequence set forth in SEQ ID NO: 40; and

[0494] The mRNA comprising a polynucleotide encoding HSV-2 glycoprotein E or an immunogenic fragment thereof linked to a heterologous signal peptide comprises one or more of: (1) RNA corresponding to the DNA of the nucleotide sequence set forth in SEQ ID NO: 27; and (2) RNA corresponding to a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 27 and that encodes an HSV-2 glycoprotein E immunogenic fragment having the amino acid sequence set forth in SEQ ID NO: 41.

[0495] In some embodiments, the pharmaceutical composition described above comprises, in addition to the nucleic acid comprising a polynucleotide encoding HSV glycoprotein D or an immunogenic fragment thereof, HSV glycoprotein C or an immunogenic fragment thereof, HSV glycoprotein E or an immunogenic fragment thereof linked to a signal peptide, a nucleic acid comprising a polynucleotide encoding another protein or an immunogenic fragment thereof.

[0496] In some embodiments, the pharmaceutical composition described above comprises, in addition to the nucleic acid comprising a polynucleotide encoding HSV glycoprotein or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding a protein or polypeptide of another virus or of HSV other than the HSV glycoprotein or an immunogenic fragment thereof that can serve as an immunogen. Of course, the other virus can be one virus subtype, or multiple viruses or multiple virus subtypes.

[0497] In some embodiments, the pharmaceutical composition described above is a nucleic acid vaccine or a protein vaccine.

[0498] In some embodiments, the pharmaceutical composition described above is a concatenated nucleic acid vaccine or a concatenated protein vaccine.

[0499] In some embodiments, the pharmaceutical composition described above is a multivalent vaccine.

[0500] In some embodiments, the pharmaceutical composition described above is an HSV vaccine.

[0501] In some embodiments, the pharmaceutical composition described above is a nucleic acid vaccine.

[0502] In some embodiments, the nucleic acid vaccine is an mRNA vaccine.

[0503] In some embodiments, the nucleic acid vaccine is a concatenated nucleic acid vaccine.

[0504] In some embodiments, the nucleic acid vaccine does not include an adjuvant.

[0505] In some embodiments, the nucleic acid vaccine further includes an adjuvant.

[0506] It can be understood that the dosage form of the nucleic acid vaccine is not particularly limited.

[0507] In some embodiments, the vaccine is an mRNA vaccine, and the dosage form of the vaccine is nasal, tracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0508] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of reducing the probability of weight loss and death caused by HSV infection.

[0509] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of reducing the titer of HSV virus after HSV infection.

[0510] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of reducing the morbidity after HSV infection.

[0511] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of improving the clinical symptoms after HSV infection.

[0512] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of reducing the copy number of HSV in dorsal root ganglion.

[0513] In some embodiments, the pharmaceutical composition (e.g., mRNA vaccine) of the present disclosure is capable of increasing the neutralizing antibody titer of HSV in serum.

[0514] In some embodiments, the above pharmaceutical composition is used for preventing or treating HSV infection.

[0515] In some embodiments, the above pharmaceutical composition is used for preventing or treating HSV-2 infection.

[0516] In some embodiments, the HSV infection is a primary HSV infection or reactivation of latent HSV infection.

[0517] In some embodiments, the HSV infection includes HSV neonatal infection or genital HSV infection.

[0518] In some embodiments, the above pharmaceutical composition is used for preventing or treating one or more of the following diseases caused by HSV infection: herpetic meningomyelitis, spinal cord radiculopathy, and pelvic inflammation.

[0519] In some embodiments, the above pharmaceutical composition is used for preventing or treating oral herpes and / or genital herpes.

[0520] In some embodiments, the pharmaceutical composition described above is used for preventing or treating genital herpes.

[0521] VII. Methods of Prevention or Treatment

[0522] The present disclosure also provides use of the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the protein or polypeptide encoded by the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments in the manufacture of a medicament for preventing or treating HSV infection.

[0523] The present disclosure also provides a method for preventing or treating HSV infection, comprising administering to a subject the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the protein or polypeptide encoded by the nucleic acid of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, the nucleic acid composition of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.

[0524] In some embodiments, the HSV infection comprises HSV-2 infection.

[0525] In some embodiments, the HSV infection comprises a primary HSV infection or reactivation of latent HSV infection.

[0526] In some embodiments, the HSV infection comprises a primary HSV-2 infection or reactivation of latent HSV-2 infection.

[0527] In some embodiments, the HSV infection comprises HSV neonatal infection or genital HSV infection.

[0528] In some embodiments, the HSV infection comprises oral HSV infection.

[0529] In some embodiments, the method described above is capable of preventing primary HSV infection, preventing reactivation of latent HSV infection, preventing HSV replication, shortening the duration of HSV infection in a subject, reducing the number of replication-competent HSV in a subject, reducing the number of cells containing HSV genome in a subject, or increasing the neutralizing antibody titer of HSV in serum of a subject.

[0530] The present disclosure also provides a method for preventing or treating herpes labialis or genital herpes, the method comprising administering to a subject the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the protein or polypeptide encoded by the nucleic acid of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, the nucleic acid composition of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.

[0531] The present disclosure also provides a method for preventing or treating herpes labialis or genital herpes, the method comprising administering to a subject the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the protein or polypeptide encoded by the nucleic acid of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, the nucleic acid composition of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.

[0532] In addition, the present disclosure also provides a method of eliciting an immune response against HSV in a subject. In some embodiments, the method comprises administering to a subject the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the protein or polypeptide encoded by the nucleic acid of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, the nucleic acid composition of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments.

[0533] In some embodiments, the subject of any of the above methods has an HSV infection or has been exposed to HSV.

[0534] In some embodiments, the subject of any of the above methods is a mammal. For example, a human, a non-human primate (e.g., an ape, a chimpanzee, a monkey, and an orangutan), a domesticated animal (e.g., a dog, a cat, and a livestock animal (e.g., a horse, a cow, a pig, a sheep, and a goat)), or another mammal. Other mammals include, but are not limited to, a mouse, a rat, a guinea pig, a rabbit, a hamster, and the like. In an alternative specific example, the subject is a human.

[0535] In some embodiments, any of the above methods comprise administering to the subject the nucleic acid, genetically engineered vector, nucleic acid composition, protein or polypeptide, RNA, immunogen or vaccine (e.g., mRNA vaccine) of any of the above embodiments at a dose level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg once, twice, or more than twice.

[0536] In some embodiments, any of the above methods comprise administering to the subject the nucleic acid, genetically engineered vector, nucleic acid composition, protein or polypeptide, RNA, immunogen or vaccine (e.g., mRNA vaccine) of any of the above embodiments at a total dose of 0.0100 mg to 1.0 mg or at a dose level sufficient to deliver the total dose once, twice, or more than twice.

[0537] In some embodiments, any of the above methods comprise administering to the subject the nucleic acid, genetically engineered vector, nucleic acid composition, protein or polypeptide, RNA, immunogen or vaccine (e.g., mRNA vaccine) of any of the above embodiments at a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg or at a dose level sufficient to deliver the total dose twice (e.g., on day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later). Higher and lower administration doses and frequencies are contemplated by the present disclosure. For example, the above vaccines (e.g., mRNA vaccines) can be administered three or four times.

[0538] VIII. Assay reagent or kit

[0539] The present disclosure also provides use of the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the polypeptide or protein encoded by the nucleic acid of any of the above embodiments, the immunogen of any of the above embodiments, the RNA of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the delivery vector of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments in the manufacture of an assay reagent or kit for HSV glycoprotein binding antibody.

[0540] Further, the present disclosure also provides an assay reagent or kit for HSV glycoprotein binding antibody, comprising the protein or polypeptide encoded by the nucleic acid of any of the above embodiments or the immunogen of any of the above embodiments.

[0541] In addition, the present disclosure also provides a method for detecting or isolating HSV glycoprotein binding antibody in a subject, comprising: providing an effective amount of the protein or polypeptide encoded by the nucleic acid of any of the above embodiments or the immunogen of any of the above embodiments; contacting a biological sample from the subject with the HSV glycoprotein under conditions sufficient to form an immunocomplex of HSV glycoprotein and HSV glycoprotein binding antibody; and detecting the immunocomplex, thereby detecting or isolating the HSV glycoprotein binding antibody in the subject. Examples

[0542] In order to make the objects and technical schemes of the present disclosure clearer, the following will be described in detail in combination with specific examples. Obviously, the described examples are only a part of the examples of the present disclosure, rather than all the examples. Based on the examples in the present disclosure, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present disclosure. In the examples, reagents and instruments are used as conventional selection in the art, unless otherwise specified. The experimental methods not specified in the examples are implemented according to conventional conditions, for example, the conditions described in the literature, books or the methods recommended by the manufacturer.

[0543] Example 1 Construction of engineering plasmid

[0544] According to Table 1, the DNA sequences corresponding to the 5'-UTR, coding region, 3'-UTR and poly(A) tail of each mRNA were synthesized and introduced into the Luciferase-pcDNA3 plasmid (Addgene, plasmid number #18964) respectively by subcloning technology (for example, subcloning technology based on PCR technology and restriction endonuclease digestion or In-fusion technology), so as to construct the engineering plasmid corresponding to each mRNA in Table 1.

[0545] Preparation of mRNA encoding HSV-2 glycoprotein immunogenic fragment connected with heterologous signal peptide

[0546] The method for synthesizing each mRNA includes but is not limited to the following steps:

[0547] 1. Extract the mRNA corresponding to the engineered plasmid prepared in Example 1, and ensure that the supercoiling rate of the plasmid is more than 85%.

[0548] 2. Linearize the plasmid by enzyme digestion, and then recover and purify it using a DNA fragment purification recovery kit (Takara, 9761) and phenol chloroform extraction purification (to remove RNase, proteins, etc.).

[0549] 3. Perform in vitro transcription on the linearized plasmid of step 2 using an IVT reaction.

[0550] 4. Purify the in vitro transcription product using Hieff RNA Cleaner (YEASEN, 12602ES56).

[0551] 5. Cap reaction

[0552] mRNA Cap1 type cap reaction:

[0553] Cap1 type cap structure and reaction principle:

[0554] pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi

[0555] ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi

[0556] G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc

[0557] m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2’-O]N1(pN)x-OH(3')+AdoHyc

[0558] 5‘-Cap1 type cap structure:

[0559] cap G 1 G 2 = m 7 G-5'-ppp-5'-Gm 2 '-3'-p-[m7 = 7-CH3; m 2 = 2'-O-CH3; -ppp- = -PO2H-O-PO2H-O-PO2H)-; -p- = -PO2H-].

[0560] A mixture of 58 pL of an aqueous solution of mRNA (125 pmol capless mRNA plus water to make up to 58 pL) was pre-warmed at 65 °C for 5 min, then mixed with a mixture containing 10 pL of 10x Capping Buffer, 10 pL of 10 mM GTP, 1.88 pL of 32 mM SAM, 0.187 pL of RNase Inhibitor, 5 pL of 2'-O-Methyltransferase (50 U / pL), and 15 pL of ScriptCap Capping Enzyme (10 U / pL), and incubated at 42 °C for 1 h. The above reagents for capping reaction were purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0561] 6. Purification of the capped product:

[0562] The capped product was purified using Hieff RNA Cleaner (YEASEN, 12602ES56) to obtain mRNA encoding the HSV-2 glycoprotein fragment linked with a heterologous signal peptide, which contains a Cap1 type cap structure, 5'-UTR, 3'-UTR, poly(A) tail, and all uridines are N1-methyl pseudouridines.

[0563] Example 3 Preparation of mRNA-encapsulated lipid nanoparticles

[0564] (1) mRNA-encapsulated lipid nanoparticles (i.e., crude product of vaccine A2) encapsulating mRNA numbered 001, 002, and 003 (each mRNA has the same content) were prepared using a microsyringe and a microfluidic chip (SN.000100) at a water phase speed of 9 mL / min and an alcohol phase speed of 3 mL / min. The mRNA numbered 001, 002, and 003 was prepared according to the method shown in Example 2; the water phase of the mRNA numbered 001, 002, and 003-encapsulated lipid nanoparticles was an acetic acid-sodium acetate solution (pH 5.0) containing mRNA numbered 001, 002, and 003 in Table 1, and the alcohol phase was an ethanol solution containing compound (IV-1-1), DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound (IV-1-1), DSPC, cholesterol, and DMG-PEG2000 was 49.5:10:39:1.5.

[0565] (3) Dialysis:

[0566] a. Preparation of dialysis solution 1 (20 mM Tris solution): Tris 1.83 g, Tris-HCl 7.05 g, 3 L distilled water, mix well to obtain 20 mM Tris solution.

[0567] b. Preparation of dialysis solution 2 (20 mM Tris + 8% (m / V) sucrose solution): Tris 1.83 g, Tris-HCl 7.05 g, 240 g sucrose, 3 L distilled water, mix well to obtain (20 mM Tris + 8% (m / V) sucrose solution.

[0568] c. Dialysis: The obtained crude product was loaded into a dialysis bag, immersed in a beaker containing 3 L of dialysis solution 1, the beaker was wrapped with aluminum foil and dialyzed at room temperature at 300 rpm for 1.5 h, then replaced with dialysis solution 2 and continued dialysis at room temperature for 1.5 h. The preparation sample after dialysis was filtered with a 0.22 μm disposable filter membrane to prepare mRNA-lipid nanoparticles encapsulating mRNA numbered 001, 002 and 003, wherein the total concentration of the three mRNAs in the mRNA-lipid nanoparticles encapsulating mRNA numbered 001, 002 and 003 was 0.2 μg / μL, and the encapsulation rate was more than 90%.

[0569] Referring to the above method, mRNA-lipid nanoparticles encapsulating mRNA numbered 001, 002 and 010 (i.e. vaccine A1), mRNA-lipid nanoparticles encapsulating mRNA numbered 004, 005 and 006 (i.e. vaccine A3) and mRNA-lipid nanoparticles encapsulating mRNA numbered 007, 008 and 009 (i.e. vaccine A4) were prepared, wherein the alcohol phase of vaccine A1 and vaccine A3 was the same as that of vaccine A2, and the alcohol phase of vaccine A4 was an ethanol solution containing ALC-0315, DSPC, cholesterol and DMG-PEG2000, wherein the molar ratio of ALC-0315, DSPC, cholesterol and DMG-PEG2000 was 49.5:10:39:1.5. At the same time, blank lipid nanoparticles (blank LNP) were prepared with an aqueous phase containing no mRNA and the same alcohol phase as vaccine A2.

[0570] Example 4 Mouse immunogenicity experiment of HSV-2 mRNA vaccine

[0571] 1. Preparation of immune serum and spleen lymphocytes

[0572] BALB / c / mice were randomly divided into 5 groups, namely: blank LNP group, vaccine A1 group, vaccine A2 group, vaccine A3 group and vaccine A4 group, wherein the blank LNP group was injected with blank LNP, the vaccine A1 group was injected with vaccine A1 (10 μg per mouse in terms of mRNA mass), and the others were the same, and vaccines A1-A4 and blank LNP were from Example 3. The mice were injected intramuscularly twice, and the first injection time was recorded as day 0 (D0), and the second injection time was day 28 (D 28 ). 28 ) and day 84 (D 84 ) after the first injection. Non-anticoagulant tubes were used to collect the blood, and the collected blood was placed on ice for 30 minutes, then centrifuged at 4°C at 3500 rpm for 10 minutes, after layering, the light yellow liquid in the upper layer was aspirated to prepare immune serum. Part of the mice collected the spleen on day 7 (D7), day 35 (D 35 ) and day 84 (D 84 ) after the first injection, and the spleen lymphocytes were isolated for subsequent experiments.

[0573] The specific preparation steps of the spleen lymphocytes include:

[0574] (1) The mice were sacrificed by cervical dislocation and immersed in 75% ethanol, and the mouse spleen was taken out in a clean bench.

[0575] (2) In a 6-well cell culture plate, 4-5 mL of mouse lymphocyte separation medium (recovered to room temperature before use and shaken well) was added to each well. The piston of the syringe was ground, and the rebound force of the cell screen was used to control the grinding force to minimize the mechanical damage to the cells.

[0576] (3) The separation solution with spleen cells was immediately transferred to a 15 mL centrifuge tube, and 10 mL of RPMI 1640 medium was slowly added to keep the liquid level clear.

[0577] (4) Centrifuge at room temperature, horizontal rotor 800g for 30 min.

[0578] (5) Aspirate the lymphocyte layer, add 10 mL of RPMI 1640 medium, and invert to wash. Centrifuge at room temperature at 250g for 10 min to collect the cells.

[0579] (6) According to the erythrocyte lysis solution instruction, the red blood cells were lysed, 3 mL of erythrocyte lysis solution was added per tube, resuspended, and placed at 4°C for 5 minutes, then 10 mL of RPMI 1640 medium was added to stop the red blood cell lysis reaction, and the cells were collected after centrifugation at 4°C at 250g for 10 min.

[0580] (7) Add 10 mL of RPMI 1640 medium to the cells collected in the previous step and wash by inverting. Collect the cells by centrifugation at 250 g for 10 min at room temperature, then resuspend the cells in culture medium and count them.

[0581] 2. True virus neutralization experiment

[0582] The HSV-2-MS-P1 virus seed (1.4 × 10⁻⁶) was used. 5.7 The titer of neutralizing antibodies in the immune serum prepared in step 1, which was inactivated by incubation at 56°C for 30 min (PFU / mL), was detected.

[0583] The operating steps include:

[0584] 1) Preparation of quality control and control samples

[0585] (1) Virus Control (VC): Prepared by mixing a certain amount of virus diluent with inactivated mixed mouse serum pre-diluted with virus inoculation solution. The mixed mouse serum is stored at -20℃ or -70℃. Its shelf life is equal to that of the single serum with the shortest shelf life in the mixed serum.

[0586] (2) Blank Control (BC): The prepared virus inoculation solution will also serve as a blank control (BC) and will not be mixed with the virus dilution solution.

[0587] (3) Positive control (PC): It is prepared by adding Mouse anti-HSV Glycoprotein D (1 mg / mL) to the inactivated mouse mixed serum.

[0588] Each board must have at least one PC hole, one VC hole, and one BC hole.

[0589] 2) Experimental steps

[0590] Day 1:

[0591] (1) Adjust the Vero cell density with complete culture medium, add 1 mL to each well of a 24-well cell culture plate, and incubate overnight at 37°C in a 5% CO2 cell culture incubator.

[0592] Day 2:

[0593] (1) Preheat the virus inoculation solution and virus maintenance solution in a water bath at 37°C.

[0594] (2) Immune serum samples were inactivated in a 56°C water bath for 30 min.

[0595] (3) Prepare sample dilution solution using inactivated mixed mouse serum.

[0596] (4) The positive control working solution was prepared with the inactivated mixed mouse serum, and then pre-diluted with the virus inoculum.

[0597] (5) Sample dilution: the immune serum sample was pre-diluted with the virus inoculum, and then continuously diluted 2-3 times with the sample diluent for 6 times.

[0598] (6) The virus was diluted with the virus inoculum.

[0599] (7) The positive control working solution or the unknown sample diluent was mixed with the virus diluent at a ratio of 1:1 in the dilution plate, and incubated at 37°C with shaking,

[0600] (8) The culture medium in the Vero cell culture plate was aspirated and discarded.

[0601] (9) The culture plate was rinsed with the virus inoculum (500 mL DMEM filtered with a 0.22 μm vacuum filter, stored at 2-8°C for 3 months).

[0602] (10) The virus inoculum was discarded, and the virus-sample mixture was added to the corresponding wells of the Vero cell culture plate according to the plate layout, with 400 μL / well of virus inoculum added to the BC wells, and incubated at 37°C in a 5% CO2 cell incubator.

[0603] (11) The liquid in the Vero cell culture plate was aspirated and discarded, and 500 μL / well of virus maintenance solution was added, and incubated at 37°C in a 5% CO2 cell incubator.

[0604] On the 4th day:

[0605] (1) The virus maintenance solution in the Vero cell plate was aspirated and discarded.

[0606] (2) Cell fixation solution was added, and incubated at room temperature.

[0607] (3) The cell fixation solution was discarded.

[0608] (4) Cell staining solution was added, and incubated at room temperature.

[0609] (5) The cell staining solution was discarded, and gently rinsed with running water.

[0610] (6) The cell plate was air-dried.

[0611] (7) Enzyme-linked spot instrument reading plate analysis.

[0612] The results are shown in Figure 1.

[0613] 3. ELISpot experiment

[0614] ELISpot assay was performed on the spleen lymphocytes obtained in step 1, and the reagents used included: RPMI Medium 1640 basic (1x), positive reference PMA + Ionomycin, ELISpot Plus Mouse IFN-gamma (HRP), peptide library based on HSV-2 gC protein (amino acid sequence as shown in SEQ ID NO: 47) (referred to as gC peptide library), peptide library based on HSV-2 gD protein (amino acid sequence as shown in SEQ ID NO: 46) (referred to as gD peptide library), and peptide library based on HSV-2 gE protein (amino acid sequence as shown in SEQ ID NO: 48) (referred to as gE peptide library).

[0615] The operation steps include:

[0616] (1) The cell suspension was prepared into 5x10 6 / mL with ELISpot special serum-free medium.

[0617] (2) Activation of pre-coated plates: 200 μL / well of RPMI 1640 medium or serum-free medium was added, and it was left at room temperature for 5-10 minutes before being removed.

[0618] (3) ELISpot plating: positive control wells: positive stimulator (PMA + Ionomycin) was added; background negative control wells: medium was added; experimental wells: corresponding peptide library (gC peptide library, gD peptide library or gE peptide library) was added.

[0619] (4) Incubation: after all samples and stimulators were added, the plate cover was covered. It was placed in a 37°C, 5% CO2 incubator for 22h.

[0620] (5) Washing the plate: the liquid in the wells was shaken out, 260 μL / well of PBS was added, the liquid in the wells was discarded, and the above steps were repeated 5 times, and each time the plate was dried on a water-absorbing paper.

[0621] (6) Detection antibody incubation: dilute the detection antibody (1:1000) to PBS-0.5% FCS, 100 μL / well. Incubate at room temperature for 2h. Wash the plate: repeat step (5).

[0622] (7) Streptavidin-HRP incubation: dilute Streptavidin-HRP (1:1000) to PBS-0.5% FCS, add to each well, 100 μL / well, incubate at room temperature for 1h.

[0623] (8) Washing the plate: the liquid in the wells was shaken out, 260 μL / well of PBS working solution was added, the liquid in the wells was discarded, and the above steps were repeated 5 times, and each time the plate was dried on a water-absorbing paper.

[0624] (9) Color development: add TMB color developing solution in the kit to each well, 100 μL / well. Incubate at room temperature in the dark for 5-10 min until obvious spots appear. Choose the time to terminate color development according to the spot generation.

[0625] (10) Wash each experimental well front and back and the base with deionized water / tap water for 3-5 times to terminate color development.

[0626] (11) Place the plate in a cool and dark place at room temperature, and then close the base after natural air drying.

[0627] (12) Count the spots on the ELISpot plate and record various parameters of the spots for statistical analysis.

[0628] The results are shown in FIGS. 2A-2C.

[0629] 4. Intracellular cytokine staining

[0630] Flow cytometry was used to perform intracellular cytokine staining (ICS) on the spleen lymphocytes obtained in step 1. The reagents used included: positive reference PMA + Ionomycin, Brilliant Violet 510 TM anti-mouse CD3ε, PerCP / Cyanine5.5 anti-mouse CD4, Brilliant Violet 650 TM anti-mouse CD8a, anti-mouse IFN-γ, Cytofix / Cytoperm Fixation / Permeablization Kit, GolgiPlug, GolgiStop, Zombie NIR TM Fixable Viability Kit, TruStain FcX (anti-mouse CD16 / 32) antibody, red blood cell lysate (10x), RPMI1640 medium.

[0631] The operation steps include:

[0632] (1) Prepare the spleen lymphocytes obtained in step 1 into a single cell suspension of 1x10 7 / mL. Take 200 μL of the cell suspension and spread in a U-bottom 96-well plate, and add the corresponding peptide library (gC peptide library, gD peptide library or gE peptide library), culture medium or positive stimulant, and GolgiPlug and Golgistop for co-culture for 6 h.

[0633] (2) Centrifuge the cells at 350 g at 4°C for 5 min, and transfer to a V-bottom 96-well plate.

[0634] (3) Blocking: Add 50 μL of prepared TruStain FcX (anti-mouse CD16 / 32) antibody to each well, resuspend the cells, mix and incubate on ice for 10 min in the dark, wash with 150 μL of cell staining buffer, centrifuge and remove the supernatant.

[0635] (4) Add 50 μL / well of a mixture of Zombie NlR TM , CD3 antibody, CD4 antibody and CD8 antibody to each well, incubate on ice for 30 min in the dark, wash once with 150 μL of cell staining buffer, centrifuge and remove the supernatant.

[0636] (5) Resuspend the cells thoroughly, add 100 uL of Cytofix / Cytoperm Fixation / Permeablization Kit fixation / permeation solution to each well, and incubate at 4°C for 20 min.

[0637] (6) Wash the cells twice in 1x BD Perm / Wash buffer.

[0638] (7) Add IFN-γ antibody diluted 1:100 in 1x BD Perm / Wash buffer to each well, then stain, place on ice in the dark for 30 min, wash once with 150 μL of cell staining buffer, centrifuge and remove the supernatant.

[0639] (8) Wash the cells once with 1x BD Perm / Wash buffer and resuspend with 150 μL of cell staining buffer.

[0640] (9) Flow cytometry analysis.

[0641] The results are shown in Figures 3A-3C and Figures 4A-4C.

[0642] Example 5 Guinea pig challenge experiment of HSV-2 mRNA vaccine

[0643] 1. Materials and methods

[0644] (1) Animals: 250-300 g, female Hartley guinea pigs of SPF level.

[0645] (2) Test substances: vaccine A2 of Example 3, blank LNP of Example 3 and vaccine A4 of Example.

[0646] (3) Virus: Herpes simplex virus type II (HSV-2, MS), original strain purchased from ATCC, item number VR-540, batch number 70032981.

[0647] 2. Animal experiment

[0648] (1) Definition of experimental days

[0649] Day 0 is the day of the first immunization, day -1 is the day before the first immunization, day 1 is the day after the first immunization, and so on.

[0650] (2) Grouping of animals

[0651] According to the experimental design, the animals were randomly divided into 5 groups.

[0652] (3) Immunization

[0653] The guinea pigs were treated with different test substances on experimental days 0, 28, and 56. The administration route was intramuscular injection, and the administration volume was 150 μL or 300 μL per site, a total of 2 sites (left hind leg and right hind leg), see Table 3 for details.

[0654] Table 3

[0655] (4) Virus inoculation

[0656] On experimental day 84, after the guinea pigs were anesthetized with Zoetile 50 (40 mg / kg) / xylazine hydrochloride (8 mg / kg) anesthetic (injection volume was 1 mL / kg), the virus solution was drawn up using a 1 mL syringe and a gavage needle, and the guinea pigs were inoculated with HSV-2 virus by vaginal perfusion, with an inoculum of 1.0E+06 PFU (plaque forming unit) per animal and an inoculum volume of 100 μL.

[0657] (5) Health monitoring

[0658] The animals were observed twice a week for survival, body weight, and injection site conditions before virus inoculation (days 0-84), and any abnormal symptoms were recorded. After virus inoculation (days 85-98), the animals were observed once a day for survival, body weight, body temperature, and clinical symptoms.

[0659] (6) Non-endpoint sample collection

[0660] Serum samples were collected by saphenous vein blood collection on days 0, 14, 28, 42, 56, 70, 84. Vaginal lavage fluid samples were collected on days 86 and 88 (2 and 4 days after virus inoculation).

[0661] Table 4

[0662] (7) End-point sample collection

[0663] DRG samples were collected on the day of animal death, or on days 97 and 98 of the experiment after guinea pigs were euthanized using CO2 inhalation. DRG samples were snap-frozen in dry ice and stored at -80 °C until shipment.

[0664] (8) humane end point

[0665] Any experimental animal that lost more than 20% of its pre-infection body weight or exhibited signs of moribundity would be euthanized and counted as a dead animal.

[0666] (9) experimental end point

[0667] The experimental end point was on days 97 and 98 of the experiment, when DRG samples were collected after half of the surviving guinea pigs in each group were euthanized using CO2 inhalation each day.

[0668] 3. Results

[0669] (1) Effect of mRNA vaccine on body weight of guinea pigs in the model

[0670] The results of body weight changes of guinea pigs in each group are shown in FIGS. 5A-5C.

[0671] Before viral infection (days 0-84), the body weight of guinea pigs in each group remained stable and did not show significant decrease.

[0672] After viral infection (days 84-98), the body weight of guinea pigs in the blank LNP group (group 5) showed significant decrease starting from day 89 (5 days after virus inoculation), and then continued to decrease until day 93 (9 days after inoculation), with the maximum decrease of -19.3% and -18.8%, respectively. The body weight of surviving guinea pigs recovered starting from day 94 (10 days after inoculation). The body weight of guinea pigs in other groups (groups 1-4) remained stable and did not show significant decrease.

[0673] (2) Effect of mRNA vaccine on survival rate of guinea pigs in the model

[0674] The survival rate of animals from day 84 to day 98 after infection was recorded. According to the established experimental protocol of IACUC, any experimental animal that lost more than 20% of its pre-infection body weight or exhibited signs of moribundity would be euthanized and counted as a dead animal. Animal deaths caused by non-viral infection were not counted as animal deaths. The results of survival rate of guinea pigs in each group are shown in FIG. 6, and the survival analysis of guinea pigs in each group is shown in Table 5.

[0675] Table 5 *: compared with group 5 blank LNP; median survival time was calculated in days after virus inoculation.

[0676] Group 1-4: No guinea pigs died during the experiment, and the final survival rate was 100%. Group 5 (blank LNP): Guinea pigs started to die from the 92nd day (8 days after virus inoculation), and the final animal survival rate was 12.5% (1 / 8).

[0677] (3) Effect of mRNA vaccine on body temperature of guinea pigs in the model

[0678] From the 84th to the 98th day after virus infection, the body temperature of animals was recorded once a day, and the results are shown in Figure 7.

[0679] Group 1-4: The body temperature of guinea pigs increased on the 86th day (2 days after virus inoculation), and then fluctuated within the normal range.

[0680] Group 5: The body temperature of guinea pigs increased on the 86th day, and then returned to normal; the body temperature continued to rise to 39.5°C from the 88th day (4 days after inoculation), and then gradually decreased; the body temperature of surviving guinea pigs returned to normal.

[0681] (4) Effect of mRNA vaccine on herpes incidence in the model

[0682] From the 84th to the 98th day after virus infection, the incidence of each group of animals was counted (herpes was counted as an illness), and the results are shown in Figure 8.

[0683] Group 1: 1 guinea pig developed herpes on the 89th day (5 days after virus inoculation), and the final animal incidence rate was 12.5% (1 / 8). Group 2: 1 guinea pig developed herpes on the 89th day, and the final animal incidence rate was 12.5% (1 / 8). Group 3: 1 guinea pig developed herpes on the 89th day, and the final animal incidence rate was 12.5% (1 / 8). Group 4: 1 guinea pig developed herpes on the 88th day (4 days after virus inoculation), and the final animal incidence rate was 12.5% (1 / 8). Group 5: 8 guinea pigs developed herpes on the 88th day, and the final animal incidence rate was 100% (8 / 8).

[0684] (5) Effect of mRNA vaccine on herpes score in the model

[0685] From the 84th to the 98th day after virus infection, the herpes incidence of guinea pigs was observed and recorded every day, and the results are shown in Figure 9.

[0686] Group 1: The guinea pigs reached the highest score of 0.1 on days 89-91, and the score was 0 on day 92 when the herpes disappeared. Group 2: The guinea pigs reached the highest score of 0.1 on days 89-91, and the score was 0 on day 92 when the herpes disappeared. Group 3: The guinea pigs reached the highest score of 0.1 on days 89-91, and the score was 0 on day 92 when the herpes disappeared. Group 4: The guinea pigs reached the highest score of 0.1 on days 88-92, and the score was 0 on day 93 when the herpes disappeared. Group 5: The guinea pigs had herpes on day 88, and the score was 1.9; the score reached the highest of 3.9 on day 90, and the score was 0 on day 96 when the herpes disappeared.

[0687] (6) Effect of mRNA vaccine on skin score in the model

[0688] From days 84-98 after viral infection, the skin scores of the animals in each group were observed and recorded every day, and the results are shown in FIG. 10.

[0689] Groups 1-3: The guinea pigs had no obvious abnormalities in the skin from days 84-98, and the scores were all 0. Group 4: The guinea pig had redness in the skin on day 94, and the score was 0.1; the redness disappeared on day 95, and the score was 0. Group 5: The guinea pig had symptoms in the skin from day 88, and the score was 0.9; the symptoms gradually worsened; the score reached the highest of 3.4 on day 92; then the symptoms gradually recovered, and the symptoms disappeared on day 98, and the score was 0.

[0690] (7) Effect of mRNA vaccine on clinical score in the model

[0691] From days 84-98 after viral infection, the clinical scores of the animals in each group were counted, and the clinical score was the sum of the herpes score and the skin score, and the results are shown in FIG. 11.

[0692] Group 1: The guinea pigs had clinical symptoms from day 89, and the clinical score reached the highest of 0.1 on days 89-91; the clinical symptoms disappeared on day 92. Group 2: The guinea pigs had clinical symptoms from day 89, and the clinical score reached the highest of 0.1 on days 89-91; the clinical symptoms disappeared on day 92. Group 3: The guinea pigs had clinical symptoms from day 89, and the clinical score reached the highest of 0.1 on days 89-91; the clinical symptoms disappeared on day 92. Group 4: The guinea pigs had clinical symptoms from day 88, and the clinical score reached the highest of 0.1 on days 88-92 and 94; the clinical symptoms disappeared on day 95. Group 5: The guinea pigs had clinical symptoms from day 88, and the symptoms gradually worsened; the clinical score reached the highest of 6.9 on days 91 and 92; the clinical symptoms disappeared on day 98.

[0693] In summary, the body weight of animals immunized with vaccine A2 and vaccine A4 kept stable growth, and no other obvious abnormal conditions were observed, which indicated that vaccine A2 and vaccine A4 were both well tolerated in guinea pigs.

[0694] The guinea pigs immunized with blank LNP developed infectious symptoms after virus inoculation, and the morbidity was 100%, the body weight of animals decreased significantly, the final survival rate was 12.5% (1 / 8), and the highest clinical symptom score was 6.9. The morbidity of guinea pigs immunized with vaccine A2 and vaccine A4 was both 12.5% after virus inoculation, and the survival rate of animals was 100%, and the highest clinical symptom score was both 0.1. Compared with blank LNP, vaccine A2 and vaccine A4 could both protect animals from body weight loss and death caused by virus infection, significantly reduce the morbidity of animals after infection, and significantly improve the clinical symptoms of animals, among which the duration of clinical symptoms of guinea pigs immunized with vaccine A2 was shorter than that of guinea pigs immunized with vaccine A4.

[0695] Example 6 Determination of neutralizing antibodies of serum samples by plaque reduction test

[0696] 1. Materials

[0697] The serum was from Example 5; the cell culture medium for Vero cells (ATCC, CCL-81) was DMEM + 10% FBS + 1% PS + 1% NEAA, and the experimental medium was DMEM + 2% FBS + 1% PS + 1% NEAA; the virus was HSV-2 (MS strain).

[0698] 2. Methods and procedures

[0699] (1) Inoculation of cells and preparation of serum samples to be tested

[0700] Vero cells were inoculated at 3 x 10 5 cells / well in a 12-well cell culture plate and cultured overnight in a 37°C, 5% CO2 incubator. The serum samples to be tested were mixed into one serum sample per group in advance into a new centrifuge tube and stored in a 4°C refrigerator overnight for detection the next day.

[0701] (2) Sample processing and virus incubation

[0702] The serum samples were heat inactivated at 56°C for 30 min, and then diluted with the experimental medium at a ratio of 1:40 and then 1:6, to obtain five dilutions (1:80, 1:480, 1:2880, 1:17280, and 1:103680). After the virus was thawed, the virus was diluted to 120 PFU / mL. 0.6 mL of the diluted serum and 0.6 mL of the diluted virus were incubated at 37°C for 1 hour. After the incubation, the medium in the 12-well plate was discarded, and the incubated mixture was added to the wells at 0.5 mL / well. A virus control (cells infected with the virus and incubated with the experimental medium) was also set. The plate was incubated at 37°C in a 5% CO2 incubator for 2 hours. After the incubation, the liquid was removed, and the cells were cultured with agarose-containing medium. After 3 days of culture at 37°C in a 5% CO2 incubator, the cells were fixed with 4% paraformaldehyde and stained with crystal violet.

[0703] The virus-neutralizing activity of the serum sample was represented by the plaque inhibition rate (%) of the serum on the virus, and the calculation formula was as follows:

[0704] The Prism software was used to calculate the neutralization titer of the serum. The curve fitting method was Sigmoidal dose-response (variable slope), and the formula was Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope).

[0705] The results are shown in FIG. 12. As shown in FIG. 12, compared with the blank LNP, both vaccine A2 and vaccine A4 can increase the neutralizing antibody titer in the serum after the second and third immunizations, and the neutralizing antibody titer of vaccine A2 is higher than that of vaccine A4.

[0706] Example 7: Application of plaque assay to detect the HSV-2 virus titer of the vaginal lavage sample

[0707] 1. Materials

[0708] The vaginal lavage was from Example 5, and the Vero cell information was the same as in Example 6.

[0709] 2. Experimental steps

[0710] (1) Cell inoculation: Vero cells (ATCC, CCL-81) were inoculated in a 6-well cell culture plate at a concentration of 6×10 5 The cells were cultured at 37°C in a 5% CO2 incubator overnight to obtain a monolayer of cells.

[0711] (2) Sample treatment and virus incubation

[0712] a) Sample processing: the vaginal lavage sample was melted in a 37 °C water bath and centrifuged for 20 s to obtain the supernatant, which was diluted 10 times with experimental medium and diluted at 5 gradients; b) Virus inoculation: remove the culture medium in the cell plate, add 0.5 mL of experimental medium, and add the diluted solution of 0.5 mL / well to the 6-well plate with cells and place it on the shaker for 5-10 minutes, set up the cell control. After mixing, it was placed in a 37 °C cell incubator for 2 hours to allow the virus to fully adsorb; c) Incubation and culture: remove the liquid in the experimental cell plate, and add 4 mL of 0.625% agarose medium solution to each well; after the agarose solidifies, it is placed in a 37 °C cell incubator for 3 days; d) Fixation and staining: on the third day, the cells were fixed with paraformaldehyde at room temperature for 4 h, then washed with water, and then 0.5 mL / well of 0.5% crystal violet solution was added, shaken for 15 minutes, then washed with water and dried; e) Plaque counting: scan the well plate and read the number of plaques, calculate the virus titer in the sample, and the virus titer is expressed as Log 10 (the number of plaques per mL of vaginal lavage sample), the calculation formula is as follows:

[0713] The data were statistically analyzed using Prism software, and One-way ANOVA, Dunnett's multiple comparisons test was used for statistical analysis. Compared with the blank LNP, "NS" represents P>0.05, "*" represents P<0.05, "**" represents P<0.01, "***" represents P<0.001, and "****" represents P<0.0001.

[0714] The virus titer results of the vaginal lavage collected on day 86 (2 days after infection) are shown in Figure 13. As can be seen from Figure 13, compared with the blank LNP, both vaccine A2 and vaccine A4 can significantly reduce the virus titer in the vaginal lavage sample.

[0715] Example 8 Application of qPCR to detect HSV-2 virus copy number in dorsal root ganglion samples

[0716] 1. Materials

[0717] The dorsal root ganglion was from the guinea pig of Example 5.

[0718] 2. Methods and procedures:

[0719] (1) Extraction of total DNA in guinea pig dorsal root ganglion: the test procedure refers to the instructions of QIAamp DNA Mini Kit (QIAGEN, 51304).

[0720] (2) Quantitative PCR detection of HSV-2 DNA content in guinea pig dorsal root ganglion

[0721] Standard curve configuration: 5 ng / μL pHSV-2 plasmid DNA containing HSV-2 partial sequence was diluted 58-fold as the highest point of qPCR standard curve 7 copy / μL, serially diluted by 10-fold with AE to generate standard curve range 7 to 10 copy / μL.

[0722] Quantitative PCR procedure: qPCR reaction mix was prepared as shown in Table 6, sample and standard were added, and PCR reaction was performed. Reaction condition: 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles.

[0723] Table 6

[0724] According to the qPCR standard curve, the HSV-2 virus copy number in guinea pig dorsal root ganglion samples was quantified, expressed as HSV-2 virus copy number / 100 ng dorsal root ganglion DNA. Statistical analysis was performed using One Way ANOVA, Dunnett's multiple comparisons test. "NS" means P>0.05, "*" means P<0.05, "**" means P<0.01, "***" means P<0.001, and "****" means P<0.0001 compared with blank LNP.

[0725] The results are shown in Figure 14. As shown in Figure 14, compared with blank LNP, vaccine A2 and vaccine A4 can significantly reduce the level of virus copy number in dorsal root ganglion.

[0726] In summary, in the guinea pig challenge model, vaccine A2 and vaccine A4 both have good in vivo protective efficacy against HSV-2 virus infection.

[0727] Sequence

[0728] SEQ ID NO: 1

[0729] SEQ ID NO: 2

[0730] SEQ ID NO: 3

[0731] SEQ ID NO: 4

[0732] SEQ ID NO: 5

[0733] SEQ ID NO: 6

[0734] SEQ ID NO: 7

[0735] SEQ ID NO: 8

[0736] SEQ ID NO: 9

[0737] SEQ ID NO: 10

[0738] SEQ ID NO: 11

[0739] SEQ ID NO: 12

[0740] SEQ ID NO: 13

[0741] SEQ ID NO: 14

[0742] SEQ ID NO: 15

[0743] SEQ ID NO: 16

[0744] SEQ ID NO: 17

[0745] SEQ ID NO: 18

[0746] SEQ ID NO: 19

[0747] SEQ ID NO: 20

[0748] SEQ ID NO: 21

[0749] SEQ ID NO: 22

[0750] SEQ ID NO: 23

[0751] SEQ ID NO: 24

[0752] SEQ ID NO: 25

[0753] SEQ ID NO: 26

[0754] SEQ ID NO: 27

[0755] SEQ ID NO: 28

[0756] SEQ ID NO: 29

[0757] SEQ ID NO: 30

[0758] SEQ ID NO: 31

[0759] SEQ ID NO: 32

[0760] SEQ ID NO: 33

[0761] SEQ ID NO: 34

[0762] SEQ ID NO: 35

[0763] SEQ ID NO: 36

[0764] SEQ ID NO: 37

[0765] SEQ ID NO: 38

[0766] SEQ ID NO: 39

[0767] SEQ ID NO: 40

[0768] SEQ ID NO: 41

[0769] SEQ ID NO: 42

[0770] SEQ ID NO: 43

[0771] SEQ ID NO: 44

[0772] SEQ ID NO: 45

[0773] SEQ ID NO: 46

[0774] SEQ ID NO: 47

[0775] SEQ ID NO: 48

[0776] SEQ ID NO: 49

Claims

A nucleic acid composition comprising one or more of the following nucleic acids comprising a polynucleotide encoding an HSV glycoprotein or an immunogenic fragment thereof: (1) a nucleic acid comprising a polynucleotide encoding an HSV glycoprotein D or an immunogenic fragment thereof; (2) a nucleic acid comprising a polynucleotide encoding an HSV glycoprotein C or an immunogenic fragment thereof; and (3) a nucleic acid comprising a polynucleotide encoding an HSV glycoprotein E or an immunogenic fragment thereof. The nucleic acid composition according to claim 1, comprising each independently a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein D or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein C or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding an HSV-2 glycoprotein E or an immunogenic fragment thereof, which are further respectively linked to a heterologous signal peptide. The nucleic acid composition according to claim 2, wherein the heterologous signal peptide comprises one or both of the following: an IgE signal peptide and an IgG kappa signal peptide. The nucleic acid composition according to claim 2 or 3, wherein the heterologous signal peptide is an IgG kappa signal peptide. Preferably, the amino acid sequence of the IgG kappa signal peptide is set forth in SEQ ID NO:

31. Preferably, the nucleotide sequence of the polynucleotide encoding the IgG kappa signal peptide is set forth in SEQ ID NO:

32. The nucleic acid composition according to any one of claims 1 to 4, wherein the HSV glycoprotein D immunogenic fragment comprises a truncated HSV-2 glycoprotein D retaining positions 26 to 393, positions 26 to 363, or positions 26 to 331 of HSV-2 glycoprotein D. The nucleic acid composition according to claim 5, wherein the HSV glycoprotein D immunogenic fragment comprises one of the following: (1) a truncated HSV-2 glycoprotein D having an amino acid sequence set forth in SEQ ID NO: 1, 4, or 7; and (2) a truncated HSV-2 glycoprotein D having an amino acid sequence that is at least 95% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 1, 4, or 7. The nucleic acid composition according to any one of claims 1 to 6, wherein the HSV glycoprotein C immunogenic fragment comprises a truncated HSV-2 glycoprotein C retaining positions 28 to 480, positions 28 to 468, or positions 27 to 426 of HSV-2 glycoprotein C. The nucleic acid composition according to claim 7, wherein the HSV glycoprotein C immunogenic fragment comprises one of the following: (1) a truncated HSV-2 glycoprotein C having an amino acid sequence set forth in SEQ ID NO: 2, 5, or 8; and (2) a truncated HSV-2 glycoprotein C having an amino acid sequence that is at least 95% and less than 100% identical to the amino acid sequence set forth in SEQ ID NO: 2, 5, or 8. The nucleic acid composition according to any one of claims 1 to 8, wherein the HSV glycoprotein E immunogenic fragment comprises a truncated HSV-2 glycoprotein E retaining positions 21 to 545, 24 to 548, 24 to 442, or 24 to 405 of HSV-2 glycoprotein E. The nucleic acid composition according to claim 9, wherein the HSV glycoprotein E immunogenic fragment comprises one of: (1) a truncated HSV-2 glycoprotein E having an amino acid sequence as set forth in SEQ ID NO: 3, 6, 9, or 42; and (2) a truncated HSV-2 glycoprotein E having an amino acid sequence with at least 95% and less than 100% identity to the amino acid sequence as set forth in SEQ ID NO: 3, 6, 9, or 42. The nucleic acid composition according to any one of claims 2 to 10, wherein the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 33; the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO: 34; and the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide has an amino acid sequence as set forth in SEQ ID NO:

35. The nucleic acid composition according to claim 11, wherein the nucleic acid comprising a polynucleotide encoding the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity to the nucleotide sequence as set forth in SEQ ID NO: 19 and encoding the HSV-2 glycoprotein D immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 33, or a RNA corresponding thereto; The nucleic acid composition according to claim 11, wherein the nucleic acid comprising a polynucleotide encoding the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity to the nucleotide sequence as set forth in SEQ ID NO: 20 and encoding the HSV-2 glycoprotein C immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 34, or a RNA corresponding thereto; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 21, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 21, and that encodes an amino acid sequence of a HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 35, or its corresponding RNA. The nucleic acid composition according to any one of claims 2-10, wherein the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide is set forth in SEQ ID NO: 33; the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide is set forth in SEQ ID NO: 34; and the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide is set forth in SEQ ID NO:

44. The nucleic acid composition according to claim 13, wherein the nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment linked to a heterologous signal peptide comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 19, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 19, and that encodes an amino acid sequence of a HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 33, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment linked to a heterologous signal peptide comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 20, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 20, and that encodes an amino acid sequence of a HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 34, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached thereto comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 45, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical and less than 100% identical to the nucleotide sequence as set forth in SEQ ID NO: 45 and that encodes an amino acid sequence of a HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 44, or its corresponding RNA. The nucleic acid composition of any one of claims 2-10, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO: 36; the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO: 37; and the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO:

38. The nucleic acid composition of claim 15, the nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached thereto comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 22, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical and less than 100% identical to the nucleotide sequence as set forth in SEQ ID NO: 22 and that encodes an amino acid sequence of a HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 36, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached thereto comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 23, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical and less than 100% identical to the nucleotide sequence as set forth in SEQ ID NO: 23 and that encodes an amino acid sequence of a HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 37, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached thereto comprises one of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 24, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 24, and that encodes an amino acid sequence of a HSV-2 glycoprotein E immunogenic fragment as set forth in SEQ ID NO: 38, or its corresponding RNA. The nucleic acid composition of any one of claims 2-10, the amino acid sequence of the HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO: 39; the amino acid sequence of the HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO: 40; and the amino acid sequence of the HSV-2 glycoprotein E immunogenic fragment with a heterologous signal peptide attached thereto is set forth in SEQ ID NO:

41. The nucleic acid composition of claim 17, the nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein D immunogenic fragment with a heterologous signal peptide attached thereto comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 25, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 25, and that encodes an amino acid sequence of a HSV-2 glycoprotein D immunogenic fragment as set forth in SEQ ID NO: 39, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein C immunogenic fragment with a heterologous signal peptide attached thereto comprises one or more of: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 26, or its corresponding RNA; and (2) a DNA having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but less than 100% identical, to the nucleotide sequence as set forth in SEQ ID NO: 26, and that encodes an amino acid sequence of a HSV-2 glycoprotein C immunogenic fragment as set forth in SEQ ID NO: 40, or its corresponding RNA; The nucleic acid containing a polynucleotide encoding a HSV-2 glycoprotein E immunogenic fragment linked to a heterologous signal peptide comprises one or more of the following: (1) a DNA having a nucleotide sequence as set forth in SEQ ID NO: 27, or a RNA corresponding thereto; and (2) a DNA having a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 27, and less than 100% identical, and which encodes an HSV-2 glycoprotein E immunogenic fragment having an amino acid sequence as set forth in SEQ ID NO: 41, or a RNA corresponding thereto. The nucleic acid composition of any one of claims 1-18, wherein the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof further comprises at least one of a 5'-UTR, a 3'-UTR, and a poly(A) tail. Preferably, the 5'-UTR corresponds to a DNA sequence as set forth in SEQ ID NO:

28. Preferably, the 3'-UTR corresponds to a DNA sequence as set forth in SEQ ID NO:

29. Preferably, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; preferably, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides; preferably, the nucleotides comprising the poly(A) tail comprise one or more nucleotides other than A nucleotides; more preferably, the poly(A) tail corresponds to a DNA sequence as set forth in SEQ ID NO:

30. The nucleic acid composition of any one of claims 1-19, wherein the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof is RNA. Preferably, the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof is mRNA. Preferably, the mRNA comprises a 5'-cap structure. The nucleic acid composition of any one of claims 1-20, wherein the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof is DNA; preferably, the DNA is capable of being transcribed into RNA. The nucleic acid composition of any one of claims 1-21, wherein the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof contains modified nucleotides. Preferably, the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof contains modified nucleosides. Preferably, the modified nucleosides comprise at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine. The protein encoded by the nucleic acid containing a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof in the nucleic acid composition of any one of claims 1-22. A non-natural nucleic acid, said non-natural nucleic acid being a nucleic acid of the nucleic acid composition of any one of claims 1 to 22 comprising a polynucleotide encoding a HSV glycoprotein or an immunogenic fragment thereof. A pharmaceutical composition comprising the nucleic acid composition of any one of claims 1 to 22, the protein of claim 23 or the non-natural nucleic acid of claim 24. The pharmaceutical composition according to claim 25, said nucleic acid composition or said non-natural nucleic acid being formulated in a delivery vehicle. The pharmaceutical composition according to claim 26, said delivery vehicle being a lipid nanoparticle, said nucleic acid composition or said non-natural nucleic acid being encapsulated in said lipid nanoparticle. The pharmaceutical composition according to any one of claims 25 to 27, said pharmaceutical composition being a vaccine; preferably, said pharmaceutical composition being an mRNA vaccine. Use of the nucleic acid composition of any one of claims 1 to 22, the protein of claim 23, the non-natural nucleic acid of claim 24 or the pharmaceutical composition of any one of claims 25 to 28 for the manufacture of a medicament for the prevention or treatment of a HSV infection; Preferably, said HSV infection comprises a HSV-2 infection; Preferably, said HSV infection is a primary HSV infection or a reactivation of a latent HSV infection; Preferably, said HSV infection comprises a HSV neonatal infection or a genital HSV infection.

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