Our original purpose in sequencing this collection of SIVsmm viruses was to provide a diverse set of SIV sequences to enable the design of SIV mosaic vaccine antigens (19
) to allow efficacy testing of the mosaic T cell vaccine concept in an SIV vaccine/SIV challenge animal model. Since HIV-1 itself does not infect macaques, protective-efficacy tests of the mosaic HIV-1 vaccine concept require either an SIV mosaic vaccine or the use of a SHIV (HIV Env cloned into in a SIV backbone) challenge, which is limited and only enables the study of Env vaccine antigens. The strikingly distinct evolutionary pathways of SIVsmm and HIV-1 we present here became evident during sequence analysis for the SIV mosaic design. The caveats we raise should be considered when interpreting more traditional heterologous-challenge/vaccine approaches as well as when using the SIV-mosaic strategy we were interested in testing. To assess the potential impact of distinct HIV-1 and SIVsmm evolutionary trajectories in the context of vaccines of experimental interest, we assessed, for both SIVsmm and HIV-1, the 9-mer coverage of two natural sequences and a two-sequence mosaic immunogen. For SIVsmm, we compared the common SIV vaccine/challenge strains SIVmacE660 and SIVmac239 and our newly designed SIV mosaic vaccine set against our collection of diverse natural SIVsmm isolates (the most comprehensive whole-genome sequence set currently available). For HIV-1, we compared a single natural B-clade and single natural C-clade sequences and a set of HIV-1 mosaic vaccine immunogens that are now approaching human phase I safety and immunogenicity trials against a set of HIV-1 M group isolates.
Genetic (DNA-based) distances were roughly comparable between the two viruses, excepting SIVsmm Pol, which has larger distance values (). At the protein level, however, the SIVsmm lineage is generally less diverse, and apparently under less positive selective pressure (or under greater fitness constraints), than the HIV-1 M group. Furthermore, inferred positions subject to positive selection were distributed differently among and within different proteins in SIVsmm and HIV. The differences in positive selection between HIV-1 and SIVsmm Gag were particularly striking: there are sites scattered throughout HIV-1 Gag that show recurrent selection throughout the phylogenetic tree (i.e., support for positive selection on a high proportion of branches ). In contrast, SIVsmm Gag was under substantially weaker positive selection (or stronger negative selection, i.e., greater fitness constraints) than Pol: the SIVsmm p27 capsid (equivalent to HIV-1 p24) showed very little variation at the amino acid level and no evidence of positive selection at all (). Capsid (p24/p27) is one of the more conserved proteins in both HIV and SIV, and certain HIV p24 epitopes are targets for HLA-restricted T cell responses associated with viral control and long-term survival (67
). Despite the overall conservation of HIV-1 p24, these epitopes still vary under CTL-mediated immune pressure, and it has been hypothesized that immune escape from these epitopes comes at a high fitness cost for the virus, contributing to the beneficial effect associated with particular HLAs (60
). The lack of inferred selection in SIVsmm p27 suggests either that the SIVsmm capsid protein is generally less tolerant of mutation than the HIV-1 capsid or that mangabeys lack the potent Gag-directed immune responses seen in humans (67
) and in macaques (55
). The latter explanation appears probable: although sooty mangabeys make CTL responses to SIV infection that are strong enough to drive viral escape (34
), and although Gag-directed CTL responses in particular do indeed exist in sooty mangabeys (59
), both the magnitude and breadth of CTL responses are reduced in SIVsmm-infected sooty mangabeys compared with those in HIV-infected humans (15
). Furthermore, SIVsmm-infected sooty mangabeys have lower neutralizing antibody titers than HIV-infected humans (35
) or SIVmac-infected rhesus macaques. In SIVagm-infected African green monkeys (56
), another “natural host,” anti-p27 antibody responses are also much weaker than in rhesus macaques; this may be a general feature of nonpathogenic primate lentiviral infections (28
). Finally, CD8+
T cell depletion experiments with SIVsmm-infected sooty mangabeys (6
) showed smaller effects on viral load than cognate experiments with macaques (32
) and African green monkeys (22
). These combined data suggest that immunological pressure on SIVsmm p27 is much lower than on HIV-1 p24. Therefore, the observed variation in SIVsmm more likely results from gradual accrual of mutations over time (i.e., genetic drift) than from rapid selection for immune evasion. In contrast, rapid immune escape is a major aspect of positive selection in HIV-1: most early mutations are concentrated within T cell epitopes (17
), viruses within individuals continually evolve to escape from neutralizing antibodies during chronic infection (3
), and HLA imprinting is evident in viruses circulating in different human populations (37
The HIV-1 M group has likely been evolving in the human population for on the order of 100 years (43
). The much greater age of the “natural host” epidemics in African monkeys, and in sooty mangabeys in particular (90
), is consistent with the greater extent of silent mutations we observed in SIVsmm, and it would furthermore allow the evolution of higher degrees of viral tolerance (virus/host coevolution for reduced pathogenicity). Sooty mangabeys, like African green monkeys, rarely progress to AIDS despite high-intensity SIVsmm infection; one mechanism by which tolerance is achieved in mangabeys is reduced immune activation (8
). Rates of progression to AIDS are associated with chronic immune activation (24
), and the failure to downregulate interferon-stimulated genes after acute infection appears to be restricted to the pathological infections that occur in macaques and humans (8
). In any case, the combined immunological and natural selection data argue strongly that the nonpathogenicity of SIVsmm in mangabeys is not due to a more effective immune response, and SIVsmm diversity appears to have evolved under lower immune pressure than HIV-1 diversity.
Diversity is of course a central problem in HIV vaccine design. A vaccine that protects against (or mitigates) HIV-1 infection must induce cross-reactive immunity against pandemic variants. We have proposed mosaic vaccines as a strategy for inducing sufficient numbers of T cell immune responses that cross-react with many different HIV-1 variants (19
To quantify T cell cross-reactivity, we previously distinguished between breadth and depth of T cell responses (5
), breadth referring to the summed quantities (counts) of different epitope loci recognized and depth referring to the recognition of multiple variants at individual epitope loci. In previous experimental work (70
), we assessed the cross-reactivity of vaccine-induced responses by comprehensively testing peptides derived from diverse HIV-1 isolates. We here introduce the theoretical counterpart, the concept of “isolate breadth,” meaning the proportion of viral isolates from a given population for which a vaccine candidate has potential epitope coverage above a given threshold. A vaccine immunogen (or immunogen set) with high isolate breadth will match large portions of many sequences in a target population, and hence the responses it induces are more likely to cross-react with epitopes of an infecting virus. As expected based on earlier findings, a stark contrast in isolate breadth is evident between the 2-mosaic immunogen cocktails and the single “standard strain” sequences (; see also Fig. S1 in the supplemental material). The contrast is consistent within both viruses and all four genes tested, not only in the highly variable Env and Nef genes but also in the relatively more conserved Pol and Gag. Since multiple vaccine-induced epitope responses per gene appear to be correlated with protection against stringent heterologous challenge in macaques (55
), and the potential epitope overlap of any single natural sequence with any likely infective strain is low (72
), optimized high-coverage immunogens such as mosaics are much more likely than single-sequence natural immunogens to have protective efficacy against HIV-1 exposure “in the wild.”
The importance of sufficient diversity in SIV heterologous-challenge studies must be emphasized: although SIVmac239 and SIVmac251 have been described by some authors as heterologous challenge pairs, an SIVmac251 challenge of SIVmac239-vaccinated animals (for examples, see references 14
), or vice versa, cannot be considered heterologous. SIVmac239 and SIVmac251 originated in the same individual macaque (Mm251-79) and are far more closely related than circulating HIV-1 sequences even within the same clade (); SIVmac239 is a clone isolated after additional macaque passages of SIVmac251 (11
). For a heterologous challenge to resemble the distances observed in the HIV circulating population, more distant viruses must be used as vaccine/challenge pairs (for instance, SIVmac239/SIVmac251 vaccination followed by SIVmacE660 challenge [7
]). SIVmacE660 is phylogenetically distinct from the SIVmac251/239 lineage (), and the genetic distance between the SIVmacE660 and SIVmac239/251 lineages is roughly comparable to the distances between HIV-1 strains circulating in the human population (93
); the criteria of phylogenetic distinctness and genetic distance should always be evaluated for proposed vaccine/challenge pairs.
Using natural variation from SIV-infected sooty mangabeys (1
) enables comparisons between polyvalent design strategies that attempt to more comprehensively address diversity and other, more traditional, polyvalent or monovalent natural-strain immunogens. Based on this premise, we gathered and sequenced novel SIVsmm strains, combined the results with existing data, and, using the full data from this augmented set, designed an SIVsmm polyvalent mosaic vaccine, which will be tested for efficacy in macaques (G.J.N., study in progress). Mosaic vaccines have been shown to induce greater numbers of epitope responses and enhanced cross-reactivity of responses (5
) compared to those of monovalent immunogens; the mosaic design presented here will enable us to test whether either or both of these improvements in immunological response may result in effective T cell responses to pathogenic heterologous viral challenge.
Considerable thought has been applied to evaluating the differences between the macaque and human immune responses to immunodeficiency viruses in the context of vaccine development (reviewed in reference 76
). In contrast, we are here attempting to highlight the potential importance of the viral component in assessing the applicability of the SIV/macaque model. The differences between the immune responses of both macaque and human nonnatural hosts are of course significant, but while macaques' immune responses to SIVsmm may indeed be more human-like than those of mangabeys, the diversity against which they are challenged appears to be derived from a much different set of selective pressures, and such differences may affect heterologous challenge. For instance, as we discuss above, SIVsmm capsid (p27), Pol RT, and Env gp120 appear to have evolved under low immunological pressure in sooty mangabeys, and they show reduced signatures of selection. The corresponding HIV-1 proteins, in contrast, have been subjected to intense immune pressure, and HIV-1 population diversity has adapted, to some degree, to the immunological diversity of its human host populations (67
). Vaccine-induced CTL responses in macaques (e.g., to the SIVmac CM9 Gag epitope [92
]) can be correlated with reduced viral load; furthermore, the overall number of Gag-directed CTL responses is correlated with viral control (55
), and CTL responses can control viral replication in the absence of neutralizing antibodies (87
). However, the relative benefits of a Gag-directed response against SIV in macaques (e.g., versus Nef- or Env-directed responses) might not be paralleled in a human population: whether particular benefits of particular responses to particular proteins observed in SIV/macaque heterologous-challenge models will translate to HIV in humans remains an open question. This is because the sequence diversity in SIVsmm-derived heterologous macaque challenges is not adapted to the macaque immune system, so even if the immune responses were qualitatively similar between macaques and humans, the viral diversity is not. Identical considerations apply to neutralizing antibody responses, since SIVmac Env protein diversity has largely evolved in the absence of strong antibody responses.
In short, the origin of interstrain diversity in macaque-adapted SIV strains is different from the origin of interclade and within-clade HIV diversity. HIV-1 diversifies under immune pressure and selection for rapid transmission; as we discuss above, SIVsmm appears to have diversified under reduced immune pressure, as well as long-term selection for low pathogenicity (78
). Throughout the spread of the HIV-1 pandemic, the initial low diversity of small viral founder populations has expanded dramatically (both within and outside Africa) via continuous passage in hosts with highly active immune systems. Laboratory SIVmac variants originated from sooty mangabey-adapted lineages, with a history of reduced immune pressure, and they have not been continuously passaged in a host with strong immune responses. Therefore, even if sequence diversity between two SIVsmm or SIVmac isolates arithmetically approximates the diversity between two HIV-1 isolates, it may not be functionally equivalent in terms of induced immune responses, and the degree of heterology against which a vaccine might be protective (i.e., the breadth of protection) may not be easily predicted from a macaque model.
Nonhuman primate models are nevertheless an invaluable tool for studying immune responses to HIV, and careful use of the various SIV/macaque models will advance the imperative goal of a protective HIV vaccine. However, the differences in immunology, pathogenesis, and diversity (29
), as well as the distinctive evolutionary pressures in the SIVsmm and HIV-1 M groups discussed here, should be considered when extrapolating from SIV/macaque experimental results to HIV/human vaccine applications.