In this study we analyzed four HIV-1 Env gp160 primary sequences. Env gp160 seems to contribute to T-cell depletion during HIV-1 infection and allows rapid transcytosis of the virus through CD4 negative cells such as the simple epithelial monolayers of the intestinal, rectal and endocervical epithelial barriers.12
Also, it seems involved in cell-to-cell spreading of HIV-1.13
Therefore, finding vaccines able to specifically target Env gp160 might inhibit HIV-1 infection and the related immunodeficiency. Moreover, HIV-1 Env gp160 was chosen as an experimental model for this study because of its high level of sequence variability.14
The four HIV-1 Env gp160 primary sequences were derived, respectively, from (1) a major HIV-1 lineage isolated in France (X01762, group M, subtype B, isolate BH10); (2 and 3) two minor variants found in Yaounde, the capital city of Cameroon (AJ291719 and AJ291720);15,16
and (4) an infectious molecular clone derived from a Spanish HIV-1 isolate (AJ006287, subtype B).17
In this way, we tried to compare common and rare HIV-1 sequences as well as HIV-1 sequences from different geographical areas.
Pentapeptides were used as scanning probes since modules of five to six amino acids represent minimal determinants involved in B- and T-cell immune recognition.18
As a matter of fact, already in 1939 Landsteiner and van der Scheer demonstrated that “antibodies may be formed which are specific for peptide chains consisting of five amino acid residues”.19
Also, Landsteiner and van der Scheer reported that antisera against the pentapeptide Gly-Gly-Gly-Gly-Leu distinguished between Gly-Gly-Gly-Gly-Leu and Gly-Gly-Leu-Gly-Gly, thus indicating that anti-pentapeptide antibodies show a high degree of specificity. This foundational paper has been followed by a number of reports, all congruent in defining pentapeptides as the minimum chain length for immune recognition.20–31
Definition of unique HIV-1 Env gp160 pentapeptides for non cross-reactive vaccine formulations.
As advocated by Kanduc,32–37
only vaccines based on unique antigenic peptides might guarantee no/low cross-reactivity and the highest specificity. Hence, as a first step in this study, we searched for pentapeptides unique to the viral proteins. illustrates the pentapeptide identity profile of the four HIV-1 Env gp160 sequences versus the human proteome, with the x-axis reporting viral pentapeptides sequentially overlapping by four residues, and the y-axis indicating the numbers of matches of each viral pentapeptide to the human proteome. clearly documents that, firstly, the pentapeptide identity profile to the human proteome has a wave pattern in the four viral proteins, with high similarity sequence areas alternating with those of low similarity. Secondly, almost all of the pentapeptide blocks forming the HIV-1 Env gp160 sequences are also repeatedly present in human proteins; thus, only a limited number of pentamers are unique to the viral proteins. A high degree of peptide matching persists even when hexapeptide motifs were used as probes for sequence identity scanning (data not shown).
Figure 1 Pentapeptide identity profile of four Env gp160 sequences to the human proteome. (A–D) refer to Env gp160 UniProtKB/Swiss-Prot accession: (A) P03375; (B) Q90DZ7; (C) Q8UMG1; and (D) O93024. (D) the ast erisk indicates the viral SSSGG pentapeptide, (more ...)
Quantification of the pentapeptide identity platform between the HIV-1 Env gp160 sequences and the human proteome is reported in . Around 90% of the pentapeptides forming the HIV-1 Env gp160 sequences also occur in the human proteome. Moreover, the shared HIV-1 Env gp160 pentapeptides are dispersed throughout the human proteome. Indeed, the mean number of times each shared 5-mer from the Env gp160 sequences occurs in the whole human proteome is ~11 (i.e., the number of multiple occurrences divided the number of shared viral pentapeptides). According to and , a vaccine formulation using an entire HIV-1 Env gp160 as an antigen would produce a risk of cross-reactivity with human proteins amounting to thousands of hits. Only about 10% of viral pentapeptides do not have a match in the human proteome; these represent, therefore, molecular signatures of the retroviral antigens. By being unique to the HIV-1 Env gp160 sequences, these zero similarity pentapeptides constitute peptide sets usable in pharmaceutical vaccine formulations theoretically devoid of any cross-reactivity potential.
Pentapeptide overlapping between four HIV-1 Env gp160 proteins and the human proteome
Definition of conserved HIV-1 Env gp160 pentapeptides for worldwide effective vaccine formulations.
HIV-1 is extraordinarily variable,38
and this variability represents a major obstacle to AIDS vaccine development. To solve the diversity problem, country- and isolate-specific vaccines can be considered, but even these solutions appear of doubtful efficacy in light of the extreme polymorphism shown by HIV-1.39
Moreover, they would add a further economic burden to the population to be vaccinated.
With the aim of identifying consensus sequences to be used in globally-effective vaccines, we analyzed HIV-1 Env gp160 unique pentapeptides for conserved sequences. That is, the four HIV-1 Env gp160 sequences were aligned and the common unique viral 5-mers were singled out.
As visualized in , alignment of the four HIV-1 Env gp160 sequences reveals a high level of conservation on the whole. Indeed, only 15 pentapeptides are both unique to the virus and possess a conserved sequence. These pentapeptides are: GMLMI, WVTVY, TLFCA, LFCAS, LKPCV, KPCVK, IPIHY, PIHYC, HYCAP, YCAPA, SFNCG, NCGGE, GEFFY, VWGIK and HIPRR.
Figure 2 Sequence alignment of the four Env gp160 sequences under study. First residue of pentapeptides unique to the viral sequences and not found in human proteins is given red. Pentapeptides unique to the viral sequences and conserved among the four Env gp160 (more ...)
These 15 unique viral pentapeptides, absent in the human proteome and common to the four Env gp160, were used to scan the PIR database of retroviral proteomes. The purpose was to ascertain whether these pentapeptides were also present in Env gp160s from other HIV strain/group/subtype isolates. An example of the data is reported in , which describes the HIVs hosting the pentapeptide KPCVK. The HIV lists relative to the remaining 14 pentapeptides are reported in Supplemental Table 3
HIVs hosting the pentapeptide KPCVK
The data in and Supplemental Table 3
are striking. Indeed, documents that antibodies against a small peptide module formed by only five amino acid residues, i.e., the pentapeptide KPCVK, would have the potential to target 57 HIV isolates from different strains (HIV-1 and HIV-2), groups (M, O and N as well as A and B) and numerous isolates of diverse geographical origins. Also, and most importantly, antibodies specific for such a small peptide module would have no cross-reactivity, i.e., they would not induce autoimmune reactions since this sequence is not represented in the human proteome.