Truncating the gp41 Cytoplasmic Tail of Simian Immunodeficiency Virus Decreases Sensitivity to Neutralizing Antibodies without Increasing the Envelope Content of Virions.

An incomplete understanding of native human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins (Envs) impedes the development of structural models of Env and vaccine design. This shortcoming is due in part to the low number of Env trimers on virus particles. For SIV, this low expression level can be counteracted by truncating the cytoplasmic tail (CT) of Env. CT truncation has been shown to increase Env incorporation into the virion and is commonly used in vaccine and imaging studies, but its effects on viral antigenicity have not been fully elucidated. To study the effects of a CT truncation of Env in viruses in similar genetic contexts, we introduced stop codons into the CT of a SIVsmE660 molecular clone and two neutralizing antibody (NAb) escape variants. These viruses shared 98% sequence identity in Env but were characterized as either tier 1 (sensitive to neutralization), tier 2 (moderately resistant to neutralization), or tier 3 (resistant to neutralization). However, the introduction of premature stop codons in Env at position Q741/Q742 converted all three transfection-derived viruses to a tier 3-like phenotype, and these viruses were uniformly resistant to neutralization by sera from infected macaques and monoclonal antibodies (MAbs). These changes in neutralization sensitivity were not accompanied by an increase in either the virion Env content of infection-derived viruses or the infectivity of transfection-derived viruses in human cells, suggesting that CT mutations may result in global changes to the Env conformation. Our results demonstrate that some CT truncations can affect viral antigenicity and, as such, may not be suitable surrogate models of native HIV/SIV Env.IMPORTANCE Modifications to the SIV envelope protein (Env) are commonly used in structural and vaccine studies to stabilize and increase the expression of Env, often without consideration of effects on antigenicity. One such widespread modification is the truncation of the Env C-terminal tail. Here, we studied the effects of a particular cytoplasmic tail truncation in three SIVsm strains that have highly similar Env sequences but exhibit different sensitivities to neutralizing antibodies. After truncation of the Env CT, these viruses were all very resistant to neutralization by sera from infected macaques and monoclonal antibodies. The viruses with a truncated Env CT also did not exhibit the desired and typical increase in Env expression. These results underscore the importance of carefully evaluating the use of truncated Env as a model in HIV/SIV vaccine and imaging studies and of the continued need to find better models of native Env that contain fewer modifications.

Adoptive Transfer of Engineered Rhesus Simian Immunodeficiency Virus-Specific CD8+ T Cells Reduces the Number of Transmitted/Founder Viruses Established in Rhesus Macaques.

AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCE: The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.

Generation and characterization of a SIVmac239 clone corrected at four suboptimal nucleotides.

BACKGROUND: SIVmac239 is a commonly used virus in non-human primate models of HIV transmission and pathogenesis. Previous studies identified four suboptimal nucleotides in the SIVmac239 genome, which putatively inhibit its replicative capacity. Since all four suboptimal changes revert to the optimal nucleotide consensus sequence during viral replication in vitro and in vivo, we sought to eliminate the variability of generating these mutations de novo and increase the overall consistency of viral replication by introducing the optimal nucleotides directly to the infectious molecular clone. RESULTS: Using site directed mutagenesis of the full-length/nef-open SIVmac239 clone, we reverted all four nucleotides to the consensus/optimal base to generate SIVmac239Opt and subsequently tested its infectivity and replicative capacity in vitro and in vivo. In primary and cell line cultures, we observed that the optimized virus displayed consistent modest but not statistically significant increases in replicative kinetics compared to wild type. In vivo, SIVmac239Opt replicated to high peak titers with an average of 1.2 × 10(8) viral RNA copies/ml at day 12 following intrarectal challenge, reaching set-point viremia of 1.2 × 10(6) viral RNA copies/ml by day 28. Although the peak and set point viremia means were not statistically different from the original "wild type" SIVmac239, viral load variation at set point was greater for SIVmac239WT compared to SIVmac239Opt (p = 0.0015) demonstrating a greater consistency of the optimized virus. Synonymous mutations were added to the integrase gene of SIVmac239Opt to generate a molecular tag consisting of ten genetically distinguishable viral variants referred to as SIVmac239OptX (Del Prete et al., J Virol. doi: 10.1128/JVI.01026-14 , 2014). Replication dynamics in vitro of these optimized clones were not statistically different from the parental clones. Interestingly, the consistently observed rapid reversion of the primer binding site suboptimal nucleotide is not due to viral RT error but is changed post-integration of a mismatched base via host proofreading mechanisms. CONCLUSIONS: Overall, our results demonstrate that SIVmac239Opt is a functional alternative to parental SIVmac239 with marginally faster replication dynamics and with increased replication uniformity providing a more consistent and reproducible infection model in nonhuman primates.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15Rα cytokine compared to IL-15 monomer.

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

Comparative characterization of transfection- and infection-derived simian immunodeficiency virus challenge stocks for in vivo nonhuman primate studies.

Simian immunodeficiency virus (SIV) stocks for in vivo nonhuman primate models of AIDS are typically generated by transfection of 293T cells with molecularly cloned viral genomes or by expansion in productively infected T cells. Although titers of stocks are determined for infectivity in vitro prior to in vivo inoculation, virus production methods may differentially affect stock features that are not routinely analyzed but may impact in vivo infectivity, mucosal transmissibility, and early infection events. We performed a detailed analysis of nine SIV stocks, comprising five infection-derived SIVmac251 viral swarm stocks and paired infection- and transfected-293T-cell-derived stocks of both SIVmac239 and SIVmac766. Representative stocks were evaluated for (i) virus content, (ii) infectious titer, (iii) sequence diversity and polymorphism frequency by single-genome amplification and 454 pyrosequencing, (iv) virion-associated Env content, and (v) cytokine and chemokine content by 36-plex Luminex analysis. Regardless of production method, all stocks had comparable particle/infectivity ratios, with the transfected-293T stocks possessing the highest overall virus content and infectivity titers despite containing markedly lower levels of virion-associated Env than infection-derived viruses. Transfected-293T stocks also contained fewer and lower levels of cytokines and chemokines than infection-derived stocks, which had elevated levels of multiple analytes, with substantial variability among stocks. Sequencing of the infection-derived SIVmac251 stocks revealed variable levels of viral diversity between stocks, with evidence of stock-specific selection and expansion of unique viral lineages. These analyses suggest that there may be underappreciated features of SIV in vivo challenge stocks with the potential to impact early infection events, which may merit consideration when selecting virus stocks for in vivo studies.

Restricted replication of xenotropic murine leukemia virus-related virus in pigtailed macaques.

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >10(10) RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.

Long-acting lenacapavir protects macaques against intravenous challenge with simian-tropic HIV.

BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.

Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.

Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen.

Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-associated envelope glycoprotein (Env) spikes. The same bnAbs also bind to recombinant, soluble trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clinical grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native versus recombinant Env glycosylation will guide vaccine design and manufacturing programs.

HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease.

HIV-1 virions are highly enriched in cholesterol relative to the cellular plasma membrane. We recently reported that a cholesterol-binding compound, amphotericin B methyl ester (AME), blocks HIV-1 entry and that single amino acid substitutions in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 confer resistance to AME. In this study, we defined the mechanism of resistance to AME. We observed that the gp41 in AME-resistant virions is substantially smaller than wild-type gp41. Remarkably, we found that this shift in gp41 size is due to cleavage of the gp41 cytoplasmic tail by the viral protease. We mapped the protease-mediated cleavage to two sites in the cytoplasmic tail and showed that gp41 truncations in this region also confer AME resistance. Thus, to escape the inhibitory effects of AME, HIV-1 evolved a mechanism of protease-mediated envelope glycoprotein cleavage used by several other retroviruses to activate envelope glycoprotein fusogenicity. In contrast to the mechanism of AME resistance observed for HIV-1, we demonstrate that simian immunodeficiency virus can escape from AME via the introduction of premature termination codons in the gp41 cytoplasmic tail coding region. These findings demonstrate that in human T cell lines, HIV-1 and simian immunodeficiency virus can evolve distinct strategies for evading AME, reflecting their differential requirements for the gp41 cytoplasmic tail in virus replication. These data reveal that HIV-1 can escape from an inhibitor of viral entry by acquiring mutations that cause the cytoplasmic tail of gp41 to be cleaved by the viral protease.

Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions.

A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication.

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

HIV-1 envelope pseudotyped viral vectors and infectious molecular clones expressing the same envelope glycoprotein have a similar neutralization phenotype, but culture in peripheral blood mononuclear cells is associated with decreased neutralization sensitivity.

Recombinant lentiviral vectors pseudotyped with heterologous HIV-1 envelope glycoproteins allow rapid and accurate measurement of antibody-mediated HIV-1 neutralization. However, the neutralization phenotypes of envelope pseudoviruses have not been directly compared to isogenic replication competent HIV-1. We produced pseudoviruses expressing three different HIV-1 envelope glycoproteins and subcloned the same three env genes into a replication competent NL4-3 molecular clone. For each of the antibodies tested, the neutralization dose-response curves of pseudoviruses and corresponding replication competent viruses were similar. Thus, envelope pseudoviruses can be used to study the anti-HIV-1 neutralizing antibody response. A single passage of replication competent virus derived from 293T cells through peripheral blood mononuclear cells (PBMC) caused a substantial decrease in sensitivity to neutralizing antibodies. This was associated with an increase in average virion envelope glycoprotein content of the PBMC-derived virus. Replication competent HIV-1 and isogenic envelope pseudoviruses have similar neutralization characteristics, but passage into PBMC is associated with decreased sensitivity to neutralization.