Selective expansion of viral variants following experimental transmission of a reconstituted feline immunodeficiency virus quasispecies

Following long-term infection with virus derived from the pathogenic GL8 molecular clone of feline immunodeficiency virus (FIV), a range of viral variants emerged with distinct modes of interaction with the viral receptors CD134 and CXCR4, and sensitivities to neutralizing antibodies. In order to assess whether this viral diversity would be maintained following subsequent transmission, a synthetic quasispecies was reconstituted comprising molecular clones bearing envs from six viral variants and its replicative capacity compared in vivo with a clonal preparation of the parent virus. Infection with either clonal (Group 1) or diverse (Group 2) challenge viruses, resulted in a reduction in CD4+ lymphocytes and an increase in CD8+ lymphocytes. Proviral loads were similar in both study groups, peaking by 10 weeks post-infection, a higher plateau (set-point) being achieved and maintained in study Group 1. Marked differences in the ability of individual viral variants to replicate were noted in Group 2; those most similar to GL8 achieved higher viral loads while variants such as the chimaeras bearing the B14 and B28 Envs grew less well. The defective replication of these variants was not due to suppression by the humoral immune response as virus neutralising antibodies were not elicited within the study period. Similarly, although potent cellular immune responses were detected against determinants in Env, no qualitative differences were revealed between animals infected with either the clonal or the diverse inocula. However, in vitro studies indicated that the reduced replicative capacity of variants B14 and B28 in vivo was associated with altered interactions between the viruses and the viral receptor and co-receptor. The data suggest that viral variants with GL8-like characteristics have an early, replicative advantage and should provide the focus for future vaccine development

Two HIV-1 Variants Resistant to Small Molecule CCR5 Inhibitors Differ in How They Use CCR5 for Entry

HIV-1 variants resistant to small molecule CCR5 inhibitors recognize the inhibitor-CCR5 complex, while also interacting with free CCR5. The most common genetic route to resistance involves sequence changes in the gp120 V3 region, a pathway followed when the primary isolate CC1/85 was cultured with the AD101 inhibitor in vitro, creating the CC101.19 resistant variant. However, the D1/86.16 escape mutant contains no V3 changes but has three substitutions in the gp41 fusion peptide. By using CCR5 point-mutants and gp120-targeting agents, we have investigated how infectious clonal viruses derived from the parental and both resistant isolates interact with CCR5. We conclude that the V3 sequence changes in CC101.19 cl.7 create a virus with an increased dependency on interactions with the CCR5 N-terminus. Elements of the CCR5 binding site associated with the V3 region and the CD4-induced (CD4i) epitope cluster in the gp120 bridging sheet are more exposed on the native Env complex of CC101.19 cl.7, which is sensitive to neutralization via these epitopes. However, D1/86.16 cl.23 does not have an increased dependency on the CCR5 N-terminus, and its CCR5 binding site has not become more exposed. How this virus interacts with the inhibitor-CCR5 complex remains to be understood

Variable Fitness Impact of HIV-1 Escape Mutations to Cytotoxic T Lymphocyte (CTL) Response

Human lymphocyte antigen (HLA)-restricted CD8+ cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs

Resistance to the CCR5 Inhibitor 5P12-RANTES Requires a Difficult Evolution from CCR5 to CXCR4 Coreceptor Use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16–18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a “resistant” variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

<p>Abstract</p> <p>Background</p> <p>Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans <it>et al</it>., <it>JAIDS </it>2008, <b>47:</b>69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.</p> <p>Results</p> <p>Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the <it>tat </it>gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.</p> <p>Conclusions</p> <p>These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.</p

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background: The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. Results: We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. Conclusions: These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication

Limiting damage during infection:lessons from infection tolerance for novel therapeutics

The distinction between pathogen elimination and damage limitation during infection is beginning to change perspectives on infectious disease control, and has recently led to the development of novel therapies that focus on reducing the illness caused by pathogens ("damage limitation") rather than reducing pathogen burdens directly ("pathogen elimination"). While beneficial at the individual host level, the population consequences of these interventions remain unclear. To address this issue, we present a simple conceptual framework for damage limitation during infection that distinguishes between therapies that are either host-centric (pro-tolerance) or pathogen-centric (anti-virulence). We then draw on recent developments from the evolutionary ecology of disease tolerance to highlight some potential epidemiological and evolutionary responses of pathogens to medical interventions that target the symptoms of infection. Just as pathogens are known to evolve in response to antimicrobial and vaccination therapies, we caution that claims of "evolution-proof" anti-virulence interventions may be premature, and further, that in infections where virulence and transmission are linked, reducing illness without reducing pathogen burden could have non-trivial epidemiological and evolutionary consequences that require careful examination

A heterogeneous human immunodeficiency virus-like particle (VLP) formulation produced by a novel vector system

HIV: An investigatory vaccine system that mimics natural infection diversity A system has been developed that allows the generation of HIV-mimicking particles which provoke an immune response from human cells. As HIV-1 remains pandemic, vaccination represents a current global research priority for prophylaxis, and could even offer a cure. Jamie Mann, of the University of Western Ontario, Canada, and an international research group have successfully created HIV-1 virus-like particles (VLPs) designed to prime immune systems against a diverse range of viral envelope protein conformations. The VLPs produced are morphologically near-identical to the virulent ‘wild-type’ HIV, and successfully stimulated the production of IFN-γ and Granzyme B from CD4+ and CD8+ T immune cells in vitro, in cell cultures taken from HIV-infected volunteers. The VLPs appeared safe in vitro; however, further investigation will confirm their safety profile in humans. The authors are currently investigating different forms of these VLPs as potential anti-HIV therapeutics