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1.  Molecularly Tagged Simian Immunodeficiency Virus SIVmac239 Synthetic Swarm for Tracking Independent Infection Events 
Journal of Virology  2014;88(14):8077-8090.
ABSTRACT
Following mucosal human immunodeficiency virus type 1 transmission, systemic infection is established by one or only a few viral variants. Modeling single-variant, mucosal transmission in nonhuman primates using limiting-dose inoculations with a diverse simian immunodeficiency virus isolate stock may increase variability between animals since individual variants within the stock may have substantial functional differences. To decrease variability between animals while retaining the ability to enumerate transmitted/founder variants by sequence analysis, we modified the SIVmac239 clone to generate 10 unique clones that differ by two or three synonymous mutations (molecular tags). Transfection- and infection-derived virus stocks containing all 10 variants showed limited phenotypic differences in 9 of the 10 clones. Twenty-nine rhesus macaques were challenged intrarectally or intravenously with either a single dose or repeated, limiting doses of either stock. The proportion of each variant within each inoculum and in plasma from infected animals was determined by using a novel real-time single-genome amplification assay. Each animal was infected with one to five variants, the number correlating with the dose. Longitudinal sequence analysis revealed that the molecular tags are highly stable with no reversion to the parental sequence detected in >2 years of follow-up. Overall, the viral stocks are functional and mucosally transmissible and the number of variants is conveniently discernible by sequence analysis of a small amplicon. This approach should be useful for tracking individual infection events in preclinical vaccine evaluations, long-term viral reservoir establishment/clearance research, and transmission/early-event studies.
IMPORTANCE Human immunodeficiency virus type 1 transmission is established by one or only a few viral variants. Modeling of limited variant transmission in nonhuman primates with a diverse simian immunodeficiency virus isolate stock may increase the variability between animals because of functional differences in the individual variants within the stock. To decrease such variability while retaining the ability to distinguish and enumerate transmitted/founder variants by sequence analysis, we generated a viral stock with 10 sequence-identifiable but otherwise genetically identical variants. This virus was characterized in vitro and in vivo and shown to allow discrimination of distinct transmission events. This approach provides a novel nonhuman primate challenge system for the study of viral transmission, evaluation of vaccines and other prevention approaches, and characterization of viral reservoirs and strategies to target them.
doi:10.1128/JVI.01026-14
PMCID: PMC4097795  PMID: 24807714
2.  FIELD IMMOBILIATION FOR TREATMENT OF AN UNKNOWN ILLNESS IN A WILD CHIMPANZEE (PAN TROGLODYTES SCHWEINFURTHII) AT GOMBE NATIONAL PARK, TANZANIA: FINDINGS, CHALLENGES AND LESSONS LEARNED 
Primates; journal of primatology  2013;55(1):10.1007/s10329-013-0372-4.
Infectious diseases are widely presumed to be one of the greatest threats to ape conservation in the wild. Human diseases are of particular concern and the costs and benefits of human presence in protected areas with apes are regularly debated. While numerous syndromes with fatal outcomes have recently been described, precise identification of pathogens remains difficult. These diagnostic difficulties are compounded by the fact that direct veterinary intervention on wild apes is quite rare. Here we present the unique case of a wild chimpanzee at Gombe National Park who was observed with a severe illness and was subsequently examined and treated in the field. Multiple specimens were collected and tested with the aim of identifying the pathogen responsible for the illness. Our findings represent the first extensive screening of a living wild chimpanzee, yet despite our efforts, the cause and source of illness remains unknown. Nevertheless, our findings represent valuable baseline data for the ape conservation community and for comparison with other recent findings. In addition, we present the case here to demonstrate the planning required and multiple types of expertise necessary to maximize the amount of data obtained from such a rare intervention, and to provide lessons learned for future studies.
doi:10.1007/s10329-013-0372-4
PMCID: PMC3872260  PMID: 23872909
apes; health; disease; intervention; diagnostics
3.  Hypercytotoxicity and Rapid Loss of NKp44+ Innate Lymphoid Cells during Acute SIV Infection 
PLoS Pathogens  2014;10(12):e1004551.
HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs), distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI) tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-β, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.
Author Summary
HIV-1 has long been shown to deplete CD4+ T cells and disrupt barrier integrity in the gastrointestinal tract, but effects on other subpopulations of lymphocytes are less well described. A recently identified subpopulation of mucosa-restricted cells, termed innate lymphoid cells (ILCs) is thought to play critical roles in maintaining homeostasis in the gastrointestinal tract and mucosal pathogen defense. Although previous work from our laboratory and others have shown SIV infection of rhesus macaques can deplete ILCs in some parts of the gastrointestinal tract, systemic as well as kinetic effects were unclear. In this report we show that ILCs, but not classical NK cells are systemically depleted during infection and also acquire cytotoxic capabilities. Furthermore, our data is the first to indicate that this important subset of innate cells is depleted acutely, permanently, and systemically during SIV infection of rhesus macaques as a model for HIV-1 infection. Given the important role of ILCs in maintaining gut homeostasis these findings could have significant implications for the understanding and treatment of HIV-induced disease.
doi:10.1371/journal.ppat.1004551
PMCID: PMC4263758  PMID: 25503264
4.  Pathogenic Features Associated with Increased Virulence upon Simian Immunodeficiency Virus Cross-Species Transmission from Natural Hosts 
Journal of Virology  2014;88(12):6778-6792.
ABSTRACT
While simian immunodeficiency viruses (SIVs) are generally nonpathogenic in their natural hosts, dramatic increases in pathogenicity may occur upon cross-species transmission to new hosts. Deciphering the drivers of these increases in virulence is of major interest for understanding the emergence of new human immunodeficiency viruses (HIVs). We transmitted SIVsab from the sabaeus species of African green monkeys (AGMs) to pigtailed macaques (PTMs). High acute viral replication occurred in all SIVsab-infected PTMs, yet the outcome of chronic infection was highly variable, ranging from rapid progression to controlled infection, which was independent of the dynamics of acute viral replication, CD4+ T cell depletion, or preinfection levels of microbial translocation. Infection of seven PTMs with plasma collected at necropsy from a rapid-progressor PTM was consistently highly pathogenic, with high acute and chronic viral replication, massive depletion of memory CD4+ T cells, and disease progression in all PTMs. The plasma inoculum used for the serial passage did not contain adventitious bacterial or viral contaminants. Single-genome amplification showed that this inoculum was significantly more homogenous than the inoculum directly derived from AGMs, pointing to a strain selection in PTMs. In spite of similar peak plasma viral loads between the monkeys in the two passages, immune activation/inflammation levels dramatically increased in PTMs infected with the passaged virus. These results suggest that strain selection and a massive cytokine storm are major factors behind increased pathogenicity of SIV upon serial passage and adaptation of SIVs to new hosts following cross-species transmission.
IMPORTANCE We report here that upon cross-species transmission and serial passage of SIVsab from its natural host, the sabaeus African green monkey (AGM), to a new host, the pigtailed macaque (PTM), viral adaptation and increased pathogenicity involve strain selection and a massive cytokine storm. These results permit the design of strategies aimed at preventing cross-species transmission from natural hosts of SIVs to humans in areas of endemicity. Furthermore, our study describes a new animal model for SIV infection. As the outcomes of SIVsab infection in PTMs, African green monkeys, and rhesus macaques are different, the use of these systems enables comparative studies between pathogenic, nonpathogenic, and elite-controlled infections, to gain insight into the mechanisms of SIV immunodeficiency and comorbidities.
doi:10.1128/JVI.03785-13
PMCID: PMC4054382  PMID: 24696477
5.  Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques 
PLoS ONE  2014;9(10):e109678.
Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID50. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.
doi:10.1371/journal.pone.0109678
PMCID: PMC4215841  PMID: 25360552
6.  A New Approach for Monitoring Ebolavirus in Wild Great Apes 
Background
Central Africa is a “hotspot” for emerging infectious diseases (EIDs) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. Ebolavirus is suspected to have caused recent declines in resident great apes. While ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm Ebola virus disease (EVD) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure.
Methodology/Principal findings
Here we report the first successful noninvasive detection of antibodies against Ebola virus (EBOV) from wild ape feces. Using this method, we have been able to identify gorillas with antibodies to EBOV with an overall prevalence rate reaching 10% on average, demonstrating that EBOV exposure or infection is not uniformly lethal in this species. Furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to EBOV (protected from exposure by rivers as topological barriers of transmission).
Conclusions/Significance
Our new approach will contribute to a strategy to protect apes from future EBOV infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. Finally, since human EVD is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where EBOV causes case-fatality rates of up to 88%.
Author Summary
Ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in Central Africa. Improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. However, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. Additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. Here we report the first successful noninvasive detection of EBOV immunity in wild great apes, demonstrating survival in this species. This tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of EVD outbreaks in human populations.
doi:10.1371/journal.pntd.0003143
PMCID: PMC4169258  PMID: 25232832
7.  A Vaccine against CCR5 Protects a Subset of Macaques upon Intravaginal Challenge with Simian Immunodeficiency Virus SIVmac251 
Journal of Virology  2014;88(4):2011-2024.
As an alternative to targeting human immunodeficiency virus (HIV), we have developed vaccines targeting CCR5, a self-protein critically involved in HIV replication and pathogenesis. By displaying peptides derived from CCR5 at high density on the surface of virus-like particles, we can efficiently induce high-titer IgG antibodies against this self-molecule. Here, we investigated whether prophylactic immunization of rhesus macaques with a particle-based vaccine targeting two regions of macaque CCR5 could prevent or suppress vaginal infection with highly virulent SIVmac251. Twelve macaques were vaccinated with a bacteriophage Qß-based vaccine targeting macaque CCR5 (Qß.CCR5). Six control animals were immunized with the Qß platform alone. All animals immunized with Qß.CCR5 developed high-titer anti-CCR5 antibody responses. Macaques were vaginally challenged with a high dose of SIVmac251. The mean peak viral RNA levels in the vaccinated groups were 30-fold lower than in the control group (106.8 versus 108.3 copies/ml plasma). Three of the 12 vaccinated macaques dramatically suppressed simian immunodeficiency virus (SIV) replication: peak viral loads were low (103 to 104 RNA copies/ml), and SIV RNA became undetectable from 6 weeks onward. No viral RNA or DNA could be detected in colon and lymph node biopsy specimens collected 13 months after challenge. In vivo depletion of CD8+ cells failed to induce a viral rebound. However, once anti-CCR5 antibody responses had waned, the 3 animals became infected after intravaginal and/or intravenous rechallenge. In conclusion, vaccination against CCR5 was associated with dramatic suppression of virus replication in a subset (25%) of macaques. These data support further research of vaccination against CCR5 to combat HIV infection.
doi:10.1128/JVI.02447-13
PMCID: PMC3911553  PMID: 24307581
8.  Immunological and Virological Mechanisms of Vaccine-Mediated Protection Against SIV and HIV 
Nature  2013;505(7484):502-508.
Summary
A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a non-human primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV Envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two amino acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of HIV-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine- or naturally-elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials.
doi:10.1038/nature12893
PMCID: PMC3946913  PMID: 24352234
9.  Using Nonhuman Primates to Model HIV Transmission 
Current opinion in HIV and AIDS  2013;8(4):280-287.
Purpose of Review
One of the major obstacles in fully understanding HIV transmission comes from the impracticality of studying transmission in humans. Because of this encumbrance, the early phases of HIV transmission and systemic dissemination are poorly understood. In order to fully comprehend these critical steps in HIV infection, animal models must be devised to accurately reflect HIV’s mode of action. This review seeks to highlight the essential nature of modeling HIV transmission in nonhuman primates.
Recent Findings
Recently it was discovered that HIV infection is established in newly infected recipients by a single or few transmitted/founder variants. This has reshaped how animal modeling is conducted with researchers currently recapitulating a physiologically relevant, low-titer infection. Pertinent animal models have been established for the most common routes of infection, including rectal, vaginal, and penile transmission; models for intravenous and oral transmission are still in developmental stages.
Summary
These limited dose models now accurately reflect HIV transmission in humans, and provide a realistic experimental platform for vaccine development and other intervention strategies which can be used to inform vaccine development in humans. Using information obtained in NHP and human trials, it is conceivable to envision effective prevention modalities in the near future.
doi:10.1097/COH.0b013e328361cfff
PMCID: PMC3999893  PMID: 23666391
transmission; NHP models; acute infection; mucosa; SIV
10.  Adaptation of HIV-1 to Its Human Host 
Molecular biology and evolution  2007;24(8):1853-1860.
Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.
doi:10.1093/molbev/msm110
PMCID: PMC4053193  PMID: 17545188
HIV-1; SIV; matrix protein; cross-species transmission; host-specific adaptation
11.  A Single Amino Acid Mutation in the Envelope Cytoplasmic Tail Restores the Ability of an Attenuated Simian Immunodeficiency Virus Mutant To Deplete Mucosal CD4+ T Cells 
Journal of Virology  2013;87(23):13048-13052.
Disruption of the conserved motif GYxxØ in the simian immunodeficiency virus (SIV) SIVmac239 envelope (Env) cytoplasmic tail resulted in a virus (ΔGY) that exhibited a high plasma peak but uniquely failed to acutely deplete mucosal CD4+ T cells. Here, we show that ΔGY containing a flanking S727P mutation that was acquired in ΔGY-infected macaques reacquired the ability to rapidly deplete CD4+ T cells in lamina propria. This suggests that the GYxxØ motif and S727P each contribute to SIV's targeting to mucosal tissues.
doi:10.1128/JVI.02126-13
PMCID: PMC3838135  PMID: 24027336
12.  Vaccine-induced myeloid cell population dampens protective immunity to SIV 
The Journal of Clinical Investigation  2014;124(6):2538-2549.
Vaccines are largely evaluated for their ability to promote adaptive immunity, with little focus on the induction of negative immune regulators. Adjuvants facilitate and enhance vaccine-induced immune responses and have been explored for mediating protection against HIV. Using a regimen of peptide priming followed by a modified vaccinia Ankara (MVA) boost in a nonhuman primate model, we found that an SIV vaccine incorporating molecular adjuvants mediated partial protection against rectal SIVmac251 challenges. Animals treated with vaccine and multiple adjuvants exhibited a reduced viral load (VL) compared with those treated with vaccine only. Surprisingly, animals treated with adjuvant alone had reduced VLs that were comparable to or better than those of the vaccine-treated group. VL reduction was greatest in animals with the MHC class I allele Mamu-A*01 that were treated with adjuvant only and was largely dependent on CD8+ T cells. Early VLs correlated with Ki67+CCR5+CD4+ T cell frequency, while set-point VL was associated with expansion of a myeloid cell population that was phenotypically similar to myeloid-derived suppressor cells (MDSCs) and that suppressed T cell responses in vitro. MDSC expansion occurred in animals receiving vaccine and was not observed in the adjuvant-only group. Collectively, these results indicate that vaccine-induced MDSCs inhibit protective cellular immunity and suggest that preventing MDSC induction may be critical for effective AIDS vaccination.
doi:10.1172/JCI73518
PMCID: PMC4038576  PMID: 24837435
13.  Immune clearance of highly pathogenic SIV infection 
Nature  2013;502(7469):10.1038/nature12519.
Established infections with the human and simian immunodeficiency viruses (HIV, SIV) are thought to be permanent with even the most effective immune responses and anti-retroviral therapies (ART) only able to control, but not clear, these infections1–4. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that ~50% of rhesus macaques (RM) vaccinated with SIV protein-expressing Rhesus Cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviremic control of infection with highly pathogenic SIVmac2395. Here, we demonstrate that regardless of route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and hematogenous viral dissemination, and that replication-competent SIV persists in multiple sites for weeks to months. However, over time, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma or tissue-associated virus using ultrasensitive assays, and loss of T cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive RT-PCR and PCR analysis of tissues from RhCMV/SIV vector-protected RM necropsied 69–172 weeks after challenge did not detect SIV RNA or DNA over background, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million hematolymphoid cells to naïve RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T cell-mediated immune surveillance elicited and maintained by CMV vectors.
doi:10.1038/nature12519
PMCID: PMC3849456  PMID: 24025770
14.  Tracking the Luminal Exposure and Lymphatic Drainage Pathways of Intravaginal and Intrarectal Inocula Used in Nonhuman Primate Models of HIV Transmission 
PLoS ONE  2014;9(3):e92830.
Over 80% of sexual HIV-1 transmissions originate from a single viral variant, but the underlying basis for this transmission bottleneck remains to be elucidated. Nonhuman primate models of mucosal virus transmission allow opportunities to gain insight into the basis of this mucosal bottleneck. We used simulated inocula consisting of either non-infectious vital dye or contrast dye with non-invasive magnetic resonance imaging (MRI) to visualize mucosal exposure and passive lymphatic drainage patterns following vaginal and rectal exposures in Indian origin rhesus macaques. Results revealed a limited overall distance of dye coverage from the anal verge following 1 ml (n  = 8) intrarectally administered, which greatly increased with a 3 ml (n = 8) volume. Intravaginal dye exposure using 2 ml revealed complete coverage of the mucosa of the vagina and ectocervix, however dye was not detectable in the endocervix, uterus, fallopian tubes or ovaries in nuliparous sexually mature rhesus macaques (n = 9). In addition, following submucosal and intranodal injections of vital dye or MRI contrast dye in the rectum (n = 9), or distal and proximal vagina (n = 4), the lymphatic drainage pathways were identified as first the internal then common iliac chain followed by para-aortic lymph nodes. Drainage from the distal descending colon (n = 8) was via the para-colonic lymph nodes followed by the inferior mesenteric and para-aortic lymph nodes. Analysis after vaginal challenge with infectious SIVmac239 followed by euthanasia at day 3 revealed a pattern of viral dissemination consistent with the imaging results. These results provide insights into potential patterns of viral dissemination that can help guide efforts to better elucidate the earliest events of virus transmission and potential intervention strategies.
doi:10.1371/journal.pone.0092830
PMCID: PMC3965472  PMID: 24667371
15.  Exposure to MIV-150 from a High-Dose Intravaginal Ring Results in Limited Emergence of Drug Resistance Mutations in SHIV-RT Infected Rhesus Macaques 
PLoS ONE  2014;9(2):e89300.
When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.
doi:10.1371/journal.pone.0089300
PMCID: PMC3937329  PMID: 24586674
16.  Molecular identification, cloning and characterization of transmitted/founder HIV-1 subtype A, D and A/D infectious molecular clones 
Virology  2012;436(1):33-48.
We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D.
doi:10.1016/j.virol.2012.10.009
PMCID: PMC3545109  PMID: 23123038
HIV-1; transmitted/founder virus; single genome sequencing; HIV-1 transmission; HIV-1 subtype A; HIV-1 subtype D; neutralizing antibodies
17.  Heterogeneity in Neutralization Sensitivities of Viruses Comprising the Simian Immunodeficiency Virus SIVsmE660 Isolate and Vaccine Challenge Stock 
Journal of Virology  2013;87(10):5477-5492.
The sooty mangabey-derived simian immunodeficiency virus (SIV) strain E660 (SIVsmE660) is a genetically heterogeneous, pathogenic isolate that is commonly used as a vaccine challenge strain in the nonhuman primate (NHP) model of human immunodeficiency virus type 1 (HIV-1) infection. Though it is often employed to assess antibody-based vaccine strategies, its sensitivity to antibody-mediated neutralization has not been well characterized. Here, we utilize single-genome sequencing and infectivity assays to analyze the neutralization sensitivity of the uncloned SIVsmE660 isolate, individual viruses comprising the isolate, and transmitted/founder (T/F) viruses arising from low-dose mucosal inoculation of macaques with the isolate. We found that the SIVsmE660 isolate overall was highly sensitive to neutralization by SIV-infected macaque plasma samples (50% inhibitory concentration [IC50] < 10−5) and monoclonal antibodies targeting V3 (IC50 < 0.01 μg/ml), CD4-induced (IC50 < 0.1 μg/ml), CD4 binding site (IC50 ∼ 1 μg/ml), and V4 (IC50, ∼5 μg/ml) epitopes. In comparison, SIVmac251 and SIVmac239 were highly resistant to neutralization by these same antibodies. Differences in neutralization sensitivity between SIVsmE660 and SIVmac251/239 were not dependent on the cell type in which virus was produced or tested. These findings indicate that in comparison to SIVmac251/239 and primary HIV-1 viruses, SIVsmE660 generally exhibits substantially less masking of antigenically conserved Env epitopes. Interestingly, we identified a minor population of viruses (∼10%) in both the SIVsmE660 isolate and T/F viruses arising from it that were substantially more resistant (>1,000-fold) to antibody neutralization and another fraction (∼20%) that was intermediate in neutralization resistance. These findings may explain the variable natural history and variable protection afforded by heterologous Env-based vaccines in rhesus macaques challenged by high-dose versus low-dose SIVsmE660 inoculation regimens.
doi:10.1128/JVI.03419-12
PMCID: PMC3648171  PMID: 23468494
18.  Comparative Characterization of Transfection- and Infection-Derived Simian Immunodeficiency Virus Challenge Stocks for In Vivo Nonhuman Primate Studies 
Journal of Virology  2013;87(8):4584-4595.
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.
doi:10.1128/JVI.03507-12
PMCID: PMC3624367  PMID: 23408608
19.  Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIVmac251 at Challenge Exposure 
Journal of Virology  2013;87(6):3538-3548.
We used the simian immunodeficiency virus mac251 (SIVmac251) macaque model to study the effect of the dose of mucosal exposure on vaccine efficacy. We immunized macaques with a DNA prime followed by SIV gp120 protein immunization with ALVAC-SIV and gp120 in alum, and we challenged them with SIVmac251 at either a single high dose or at two repeated low-dose exposures to a 10-fold-lower dose. Infection was neither prevented nor modified following a single high-dose challenge of the immunized macaques. However, two exposures to a 10-fold-lower dose resulted in protection from SIVmac251 acquisition in 3 out of 12 macaques. The remaining animals that were infected had a modulated pathogenesis, significant downregulation of interferon responsive genes, and upregulation of genes involved in B- and T-cell responses. Thus, the choice of the experimental model greatly influences the vaccine efficacy of vaccines for human immunodeficiency virus (HIV).
doi:10.1128/JVI.02863-12
PMCID: PMC3592147  PMID: 23325681
20.  Antibodies with High Avidity to the gp120 Envelope Protein in Protection from Simian Immunodeficiency Virus SIVmac251 Acquisition in an Immunization Regimen That Mimics the RV-144 Thai Trial 
Journal of Virology  2013;87(3):1708-1719.
The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8+ T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIVmac251 that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4+ and CD8+ T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIVmac251 acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIVmac251-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIVmac251 infectivity in cells that express high levels of α4β7 integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.
doi:10.1128/JVI.02544-12
PMCID: PMC3554145  PMID: 23175374
21.  Loss of a Tyrosine-Dependent Trafficking Motif in the Simian Immunodeficiency Virus Envelope Cytoplasmic Tail Spares Mucosal CD4 Cells but Does Not Prevent Disease Progression 
Journal of Virology  2013;87(3):1528-1543.
A hallmark of pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections is the rapid and near-complete depletion of mucosal CD4+ T lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here, we evaluate in rhesus macaques a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4+ T cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation and declining peripheral blood CD4+ T cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or central nervous system tissues even in late-stage disease. Although the ΔGY mutation persisted, novel mutations evolved, including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wild-type levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.
doi:10.1128/JVI.01928-12
PMCID: PMC3554169  PMID: 23152518
22.  PATHOLOGIC LESIONS IN CHIMPANZEES (PAN TROGYLODYTES SCHWEINFURTHII) FROM GOMBE NATIONAL PARK, TANZANIA, 2004–2010 
During a population decline or disease outbreak, the true risk of specific diseases to a wild population is often difficult to determine because of a lack of baseline disease information. To better understand the risk of disease in an endangered and scientifically important population of chimpanzees (Pan trogylodytes schweinfurthii), a health monitoring program was initiated in Gombe National Park, Tanzania. As part of this health monitoring program, comprehensive necropsies with histopathology were conducted on chimpanzees (n =11; 5 male, 6 female), ranging in age from fetal to 44 yr, that were found dead between August 2004 and January 2010. In contrast to previous reports, respiratory disease was not noted as a cause of morbidity or mortality. Trauma was the most common cause of death in these 11 chimpanzees. All of the chimpanzees greater than 1 yr of age had intestinal and mesenteric parasitic granulomas associated with true strongyles consistent with Oesophagostomum spp. The relative numbers of granulomas increased with age and, in some cases, may have been a cause of weight loss and diarrhea. Simian immunodeficiency virus (SIV)cpz infection was documented in four deceased apes, all of whom exhibited varying amounts of lymphoid depletion including two females with marked CD4+ T cell loss consistent with end-stage SIVmac or human immunodeficiency virus infections. Myocardial megalokaryosis was common in chimpanzees greater than 1 mo of age; yet myocardial interstitial fibrosis, a common lesion in captive chimpanzees, was uncommon and only noted in two aged chimpanzees. These findings provide important information on causes of morbidity and mortality in wild chimpanzees, information that can be used to interpret findings during population declines and lead to better management of this population in the context of disease risk.
PMCID: PMC3693847  PMID: 22204054
Chimpanzee; pathology; Oesophagostomum spp; simian immunodeficiency virus; Gombe National Park
23.  Eastern Chimpanzees, but Not Bonobos, Represent a Simian Immunodeficiency Virus Reservoir 
Journal of Virology  2012;86(19):10776-10791.
Chimpanzees in west central Africa (Pan troglodytes troglodytes) are endemically infected with simian immunodeficiency viruses (SIVcpzPtt) that have crossed the species barrier to humans and gorillas on at least five occasions, generating pandemic and nonpandemic forms of human immunodeficiency virus type 1 (HIV-1) as well as gorilla SIV (SIVgor). Chimpanzees in east Africa (Pan troglodytes schweinfurthii) are also infected with SIVcpz; however, their viruses (SIVcpzPts) have never been found in humans. To examine whether this is due to a paucity of natural infections, we used noninvasive methods to screen wild-living eastern chimpanzees in the Democratic Republic of the Congo (DRC), Uganda, and Rwanda. We also screened bonobos (Pan paniscus) in the DRC, a species not previously tested for SIV in the wild. Fecal samples (n = 3,108) were collected at 50 field sites, tested for species and subspecies origin, and screened for SIVcpz antibodies and nucleic acids. Of 2,565 samples from eastern chimpanzees, 323 were antibody positive and 92 contained viral RNA. The antibody-positive samples represented 76 individuals from 19 field sites, all sampled north of the Congo River in an area spanning 250,000 km2. In this region, SIVcpzPts was common and widespread, with seven field sites exhibiting infection rates of 30% or greater. The overall prevalence of SIVcpzPts infection was 13.4% (95% confidence interval, 10.7% to 16.5%). In contrast, none of the 543 bonobo samples from six sites was antibody positive. All newly identified SIVcpzPts strains clustered in strict accordance to their subspecies origin; however, they exhibited considerable genetic diversity, especially in protein domains known to be under strong host selection pressure. Thus, the absence of SIVcpzPts zoonoses cannot be explained by an insufficient primate reservoir. Instead, greater adaptive hurdles may have prevented the successful colonization of humans by P. t. schweinfurthii viruses.
doi:10.1128/JVI.01498-12
PMCID: PMC3457319  PMID: 22837215
24.  Pathogenicity and Mucosal Transmissibility of the R5-Tropic Simian/Human Immunodeficiency Virus SHIVAD8 in Rhesus Macaques: Implications for Use in Vaccine Studies 
Journal of Virology  2012;86(16):8516-8526.
There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIVAD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIVAD8-CE8J, SHIVAD8-CK15, and SHIVAD8-CL98) exhibited robust replication in vivo and caused marked depletion of CD4+ T cells affecting both memory and naïve CD4+ T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIVAD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections (Pneumocystis, Candida, and Mycobacterium). A single but unique founder virus, also present in the SHIVAD8-CE8J swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIVAD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4+ T lymphocytes in vivo, and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments.
doi:10.1128/JVI.00644-12
PMCID: PMC3421705  PMID: 22647691
25.  SIVmac251 Is Inefficiently Transmitted to Rhesus Macaques by Penile Inoculation with a Single SIVenv Variant Found in Ramp-up Phase Plasma 
AIDS Research and Human Retroviruses  2011;27(12):1259-1269.
Abstract
Despite the fact that approximately half of all HIV patients acquire infection through penile exposure, there have been no recent studies of penile SIV transmission in rhesus macaques and the nature of the virus variants transmitted, target cells, and pathways of virus dissemination to systemic lymphoid tissues are not known. Single genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Studies using the SGA strategy have shown that intrarectal and intravaginal SIV transmission to macaques recapitulates key features of human HIV transmission. To date, no studies have used the SGA assay to identify transmitted/founder virus(es) in macaques infected after penile SIV exposure. Here we report that SIV can be transmitted by penile SIV exposure. However, similar exposure to a high-dose inoculum infects only about half the animals, which is about 50% less efficient transmission than occurs after vaginal SIV challenge. In addition, only a single SIV env variant established the systemic infection in all five animals that became infected after penile exposure, a result that is consistent with low incidence and few transmitted HIV variants in heterosexually infected men. Our results suggest that the penile transmission of SIVmac251 in rhesus macaques recapitulates the key features of penile HIV-1 transmission and may provide insight into host or viral factors that permit penile transmission and dissemination. Furthermore, this SIV challenge exposure route will be useful in testing vaccines and other prophylactic approaches.
doi:10.1089/aid.2011.0090
PMCID: PMC3227244  PMID: 21732792

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