HIV VACCINES AND HOW TO USE THEM

Abstract

Provided are HIV-1 fusion polypeptides, polynucleotides encoding such fusion polypeptides, vectors expressing such fusion polypeptides for use in inducing an immune response against HIV-1; pharmaceutical and immunogenic compositions and kits containing such fusion polypeptides, polynucleotides or vectors, and methods for use in treating and / or preventing HIV-1. In addition, provided are methods for constructing antiviral vaccines, including vaccines for inducing an immune response against HIV-1.

Claims

1. A computerized method for designing a fusion polypeptide that is capable of inducing an immune response against one or more viral target antigens, the method comprising: a) identifying in silico one or more regions of sequence conservation in a population of polypeptide sequences encoded by a viral gene, where the population is a population of viruses from individual patients, by performing an algorithm comprising the following steps: 1) alignment of the set of initial viral proteomic sequences with the reference sequence; 2) extraction of all unique 9-mers from viral proteomic sequences and determination of their frequency, and construction of sets of 9-mer pairs with frequencies; 3) joining pairs of 9-mers at adjacent positions of conserved region alignments that have an overlap of eight amino acids; and 4) determining the path from the first position of the 9-mer to the last position based on the largest sum of the frequencies of all 9-mers in the specified path; b) identifying in silico the two most abundant polypeptide sequences from one or more conserved regions identified in step a), and generating bivalent polypeptide segments from the conserved regions, thereby generating bivalent vaccine sequences based on the bivalent pathway of the 9-mer pair of step a) and joining two 9-mers at adjacent positions within the bivalent pathway of the 9-mer pair if they have an 8 amino acid overlap; and c) a reduction or removal of viral polypeptide 9-mers that have at least 55% (5 of 9 amino acid residues) amino acid sequence identity with a human protein; and d) an arrangement of polypeptide segments to reduce or prevent the creation of deleterious epitopes at junctions between polypeptide segments, including the reduction or elimination of binding 9-mers that bind to a particular HLA allele with a predicted IC50 value of less than about 1000 nM or having a percentile rank within the top 5% in a population of polypeptide segments where the MHC binding affinity is predicted using one or more algorithms; and e) shortening the length of the fusion polypeptide, wherein the remaining subsequences of the polypeptide segments contain epitopes (i) predicted in silico and (ii) confirmed in vitro.

2. The method of claim 1, further comprising the step of identifying subsequence variants within one or more regions of sequence conservation in a population of polypeptides of an individual patient encoded by a viral gene, for example, using deep sequencing data.

3. The method of claim 2, further comprising the step of identifying conserved regions of the polypeptide encoded by the viral gene such that at least 70% of the subsequence variants within one or more sequence conservation regions in the patient population are within bivalent polypeptide segments.

4. The method according to any one of claims 1 to 3, further comprising the step of shortening the length of the fusion polypeptide by at least 10%, wherein the remaining subsequences of the polypeptide segments comprise epitopes (i) predicted in silico and (ii) confirmed in vitro.

5. The method according to any one of claims 1 to 4, wherein the MHC binding affinity is predicted using one or more algorithms selected from NetMHC and MHCflurry.

6. A computerized method for producing a bivalent antigen, comprising the in silico construction of a set of polyvalent amino acid sequences in structurally conserved regions of a population of viral proteomic sequences using a method comprising (a) alignment of a population of viral proteomic sequences; (b) generating for each sequence in the alignment a set of 9 amino acid subsequences ("9-mers"), starting from the N-terminal amino acid, wherein each subsequence overlaps the previous subsequence by eight amino acids such that each sequence of length l in the alignment contains (l-8) 9-mers; (c) calculating the frequency for each unique 9-mer starting from position i in each sequence alignment and identifying two or more common unique 9-mers at each position; (c)(1) where the frequency is calculated as the number of times a unique 9-mer occurs at position i in the alignment divided by the total number of sequences in the alignment; (d) calculating the polyvalent conservation for each position by summing the proportion of sequences in the alignment containing any of the two or more most common unique 9-mers; (e) creating an alignment of conserved regions by subtracting sequences in the alignment that have polyvalent conservation greater than 80% or greater than 90%; (f) determining the frequency for each pair of unique 9-mers at each position in the alignment of conserved regions; (g) joining a pair of 9-mers at adjacent positions of the conserved region alignment that have an overlap of eight amino acids; (h) creating a directed acyclic graph in which each pair of 9-mers represents a node and the edges between adjacent nodes are formed from connected pairs of 9-mers at adjacent positions with the mass of each edge equal to the frequency of the underlying pair of 9-mers, adding a source node and connecting it to all nodes in the first position, adding a drain node and connecting it to all nodes in the last position, and denial of all masses; (i) finding the optimal path in a directed acyclic graph from a source node to a sink node, where the optimal path is defined as the sum of the frequencies of all pairs of 9-mers in the directed acyclic graph; (j) constructing a multivalent antigen by joining two or more 9-mers at adjacent positions in an optimal multivalent 9-mer pathway if they have an overlap of eight amino acids, thereby creating two or more sequences of joined 9-mers that together form a multivalent antigen; and (k) rearranging polypeptide segments to reduce or prevent the creation of deleterious epitopes at junctions between polypeptide segments, including reducing or removing binding 9-mers that bind to a particular HLA allele with a predicted IC50 value of less than about 1000 nM or having a percentile rank within the top 5% in a population of polypeptide segments, wherein the MHC binding affinity is predicted using one or more algorithms.

7. The method according to claim 6, wherein in step (a) the conserved regions are further determined by performing one or more of the following steps: (i) removal of segments from 9 amino acids to 35 amino acids in length; (ii) removal of segments determined to have less than 90% bivalent conservation; (iii) removal of segments that are determined to be weakly immunogenic or non-immunogenic, as demonstrated in vitro or in vivo; and / or (iv) inclusion of additional segments that are determined to be immunogenic as demonstrated in vitro or in vivo.

8. The method according to any one of claims 6-7, wherein the step of rearranging peptide segments to reduce or prevent the creation of deleterious epitopes is carried out using a method comprising one or more of an in silico HLA binding assay and a human proteome cross-recognition assay.

9. The method according to any one of claims 6 to 8, further comprising inserting a linker sequence between one or more adjacent segments.

10. The method according to any one of claims 6 to 9, wherein the method further comprises improving the bivalent antigen obtained in step (h) by removing 9-mers that have at least 55% (5 of 9 amino acid residues) amino acid sequence identity with human peptides, or that have the same T-cell receptor (TCR) as human proteins.

11. The method according to any one of claims 6 to 10, further comprising the step of identifying subsequence variants within one or more regions of sequence conservation in a population of polypeptides encoded by a viral gene, for example using deep sequencing data.

12. The method of claim 11, further comprising the step of identifying conserved regions of the polypeptide encoded by the viral gene such that at least 70% of the subsequence variants within one or more sequence conservation regions in the patient population are within bivalent polypeptide segments.

13. The method according to any one of claims 6 to 12, further comprising the step of shortening the length of the fusion polypeptide by at least 10%, wherein the remaining subsequences of the polypeptide segments comprise epitopes (i) predicted in silico and (ii) confirmed in vitro.

14. The method according to any one of claims 1 to 13, wherein one or more target viral antigens are derived from a mammalian virus, such as a human virus.

15. The method according to any one of claims 1 to 14, wherein one or more viral target antigens are derived from a virus selected from human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), herpes simplex virus (HSV), Ebola virus, Zika virus and Chikungunya virus.

16. The method according to any one of claims 1 to 15, wherein the population of viruses of individual patients is derived from a population of patients who have not received antiretroviral therapy (ART).

17. The method according to any one of claims 1 to 16, wherein the population of viruses of individual patients is derived from a population of patients who have received antiretroviral therapy (ART).

18. A fusion polypeptide obtained according to the method of any one of claims 1 to 17, wherein the fusion polypeptide elicits an immune response against a virus in a mammal.

19. The fusion polypeptide of claim 18, wherein the mammal is a human.

Images