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Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects’ cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.

Prompt identification of newly HIV-infected persons, particularly those who are most at risk of extended high viremia (EHV), allows important clinical and transmission prevention benefits. We sought to determine whether EHV could be predicted during early HIV infection (EHI) from clinical, demographic, and laboratory indicators in a large HIV-1 incidence study in Africa.Adults acquiring HIV-1 infection were enrolled in an EHI study assessing acute retroviral syndrome (ARS) symptoms and viral dynamics.Estimated date of infection (EDI) was based on a positive plasma viral load or p24 antigen test prior to seroconversion, or the mid-point between negative and positive serological tests. EHV was defined as mean untreated viral load ≥5 log10 copies/ml 130-330 days post-EDI. We used logistic regression to develop risk score algorithms for predicting EHV based on sex, age, number of ARS symptoms, and CD4 and viral load at diagnosis.Models based on the full set of five predictors had excellent performance both in the full population (c-statistic = 0.80) and when confined to persons with each of three HIV-1 subtypes (c-statistic = 0.80-0.83 within subtypes A, C, and D). Reduced models containing only 2-4 predictors performed similarly. In a risk score algorithm based on the final full-population model, predictor scores were one for male sex and enrollment CD44.9 log10 copies/ml. With a risk score cut-point of two, this algorithm was 85% sensitive (95% CI: 76%-91%) and 61% specific (55%-68%) in predicting EHV.Simple risk score algorithms can reliably identify persons with EHI in sub-Saharan Africa who are likely to sustain high viral loads if treatment is delayed. These algorithms may be useful for prioritizing intensified efforts around care linkage and retention, treatment initiation, adherence support, and partner services to optimize clinical and prevention outcomes.

HIV epidemiology informs prevention trial design and program planning. Nine clinical research centers (CRC) in sub-Saharan Africa conducted HIV observational epidemiology studies in populations at risk for HIV infection as part of an HIV prevention and vaccine trial network. Annual HIV incidence ranged from below 2% to above 10% and varied by CRC and risk group, with rates above 5% observed in Zambian men in an HIV-discordant relationship, Ugandan men from Lake Victoria fishing communities, men who have sex with men, and several cohorts of women. HIV incidence tended to fall after the first three months in the study and over calendar time. Among suspected transmission pairs, 28% of HIV infections were not from the reported partner. Volunteers with high incidence were successfully identified and enrolled into large scale cohort studies. Over a quarter of new cases in couples acquired infection from persons other than the suspected transmitting partner.

2013 WHO guidelines recommend starting ART at CD4+ T-cell counts ≤500 cells/μL. We present the T-cell counts from adult Africans with HIV shortly following transmission to their sexual partners.HIV-discordant couples in Zambia, Uganda and Rwanda were followed prospectively and received couples counseling and condoms. HIV uninfected partners were tested for HIV at least quarterly and HIV-infected partners received HIV care and referral for ART per national guidelines. Upon diagnosis of incident HIV infection in the previously HIV-uninfected partner, a blood sample was collected from both partners to measure CD4+ T-cells and perform viral linkage. The estimated date of infection (EDI) of the incident case was calculated based on testing history. EDI was unknown for suspected transmitting partners.From 2006-2011, 4,705 HIV-discordant couples were enrolled in this cohort, and 443 cases of incident HIV infection were documented. Virus linkage analysis was performed in 374 transmission pairs, and 273 (73%) transmissions were linked genetically. CD4 counts in the transmitting partner were measured a median of 56 days after EDI (mean:90.5, min:10, max:396). The median CD4 count was 339 cells/μl (mean:386.4, min:15, max:1,434), and the proportion of partners with a CD4+ T-cell count above 500/μl was 25% (95% CI:21, 31).In our cohort of discordant couples, 73% of HIV transmissions occurred within the relationship, and the transmitter CD4+ T cell count shortly after the transmission event was frequently higher than the WHO 2013 ART-initiation guidelines.

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.

Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

All genes in the TRIM6/TRIM34/TRIM5/TRIM22 locus are type I interferon inducible, with TRIM5 and TRIM22 possessing antiviral properties. Evolutionary studies involving the TRIM6/34/5/22 locus have predominantly focused on the coding sequence of the genes, finding that TRIM5 and TRIM22 have undergone high rates of both non-synonymous nucleotide replacements and in-frame insertions and deletions. We sought to understand if divergent evolutionary pressures on TRIM6/34/5/22 coding regions have selected for modifications in the non-coding regions of these genes and explore whether such non-coding changes may influence the biological function of these genes. The transcribed genomic regions, including the introns, of TRIM6, TRIM34, TRIM5, and TRIM22 from ten Haplorhini primates and one prosimian species were analyzed for transposable element content. In Haplorhini species, TRIM5 displayed an exaggerated interspecies variability, predominantly resulting from changes in the composition of transposable elements in the large first and fourth introns. Multiple lineage-specific endogenous retroviral long terminal repeats (LTRs) were identified in the first intron of TRIM5 and TRIM22. In the prosimian genome, we identified a duplication of TRIM5 with a concomitant loss of TRIM22. The transposable element content of the prosimian TRIM5 genes appears to largely represent the shared Haplorhini/prosimian ancestral state for this gene. Furthermore, we demonstrated that one such differentially fixed LTR provides for species-specific transcriptional regulation of TRIM22 in response to p53 activation. Our results identify a previously unrecognized source of species-specific variation in the antiviral TRIM genes, which can lead to alterations in their transcriptional regulation. These observations suggest that there has existed long-term pressure for exaptation of retroviral LTRs in the non-coding regions of these genes. This likely resulted from serial viral challenges and provided a mechanism for rapid alteration of transcriptional regulation. To our knowledge, this represents the first report of persistent evolutionary pressure for the capture of retroviral LTR insertions.

Acute HIV infection (prior to antibody seroconversion) represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent.Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1) Antigen positive, antibody negative (acute infection); 2) Antigen positive, antibody positive; 3) Antigen negative, antibody positive; 4) Antigen negative, antibody negative; and 5) Antigen false positive, antibody negative (HIV uninfected). A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both.Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106), 1 (2.9%) was detected by the Combo antigen component, 7 (20.6%) others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18). Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9). One false positive Combo antibody result (1/30, 3.3%) was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL.Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

BACKGROUND:Acquisition of human immunodeficiency virus type 1 (HIV-1) infection is mediated by a combination of characteristics of the infectious and the susceptible member of a transmission pair, including human behavioral and genetic factors, as well as viral fitness and tropism. Here we report on the impact of established and potential new HLA class I determinants of heterosexual HIV-1 acquisition in the HIV-1-exposed seronegative (HESN) partners of serodiscordant Zambian couples. METHODOLOGY/PRINCIPAL FINDINGS:We assessed the relationships of behavioral and clinically documented risk factors, index partner viral load, and host genetic markers to HIV-1 transmission among 568 cohabiting couples followed for at least nine months. We genotyped subjects for three classical HLA class I genes known to influence immune control of HIV-1 infection. From 1995 to December 2006, 240 HESNs seroconverted and 328 remained seronegative. In Cox proportional hazards models, HLA-A*68:02 and the B*42-C*17 haplotype in HESN partners were significantly and independently associated with faster HIV-1 acquisition (relative hazards = 1.57 and 1.55; p = 0.007 and 0.013, respectively) after controlling for other previously established contributing factors in the index partner (viral load and specific class I alleles), in the HESN partner (age, gender), or in the couple (behavioral and clinical risk score). Few if any previously implicated class I markers were associated here with the rate of acquiring infection. CONCLUSIONS/SIGNIFICANCE:A few HLA class I markers showed modest effects on acquisition of HIV-1 subtype C infection in HESN partners of discordant Zambian couples. However, the striking disparity between those few markers and the more numerous, different markers found to determine HIV-1 disease course makes it highly unlikely that, whatever the influence of class I variation on the rate of infection, the mechanism mediating that phenomenon is identical to that involved in disease control.

During untreated, chronic HIV-1 infection, plasma viral load (VL) is a relatively stable quantitative trait that has clinical and epidemiological implications. Immunogenetic research has established various human genetic factors, especially human leukocyte antigen (HLA) variants, as independent determinants of VL set-point.To identify and clarify HLA alleles that are associated with either transient or durable immune control of HIV-1 infection, we evaluated the relationships of HLA class I and class II alleles with VL among 563 seroprevalent Zambians (SPs) who were seropositive at enrollment and 221 seroconverters (SCs) who became seropositive during quarterly follow-up visits. After statistical adjustments for non-genetic factors (sex and age), two unfavorable alleles (A*3601 and DRB1*0102) were independently associated with high VL in SPs (p