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One component of the Joint United Nations Programme on HIV/AIDS (UNAIDS) goal to end the HIV/AIDS epidemic by 2030, is that 95% of all persons receiving antiretroviral therapy (ART) achieve viral suppression.† Thus, testing all HIV-positive persons for viral load (number of copies of viral RNA per mL) is a global health priority (1). CDC and other U.S. government agencies, as part of the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), together with other stakeholders, have provided technical assistance and supported the cost for multiple countries in sub-Saharan Africa to expand viral load testing as the preferred monitoring strategy for clinical response to ART. The individual and population-level benefits of ART are well understood (2). Persons receiving ART who achieve and sustain an undetectable viral load do not transmit HIV to their sex partners, thereby disrupting onward transmission (2,3). Viral load testing is a cost-effective and sustainable programmatic approach for monitoring treatment success, allowing reduced frequency of health care visits for patients who are virally suppressed (4). Viral load monitoring enables early and accurate detection of treatment failure before immunologic decline. This report describes progress on the scale-up of viral load testing in eight sub-Saharan African countries from 2013 to 2018 and examines the trajectory of improvement with viral load testing scale-up that has paralleled government commitments, sustained technical assistance, and financial resources from international donors. Viral load testing in low- and middle-income countries enables monitoring of viral load suppression at the individual and population level, which is necessary to achieve global epidemic control. Although there has been substantial achievement in improving viral load coverage for all patients receiving ART, continued engagement is needed to reach global targets.

Introduction As access to antiretroviral therapy (ART) for people with HIV (PWH) in the Republic of South Sudan (RSS) increases, viral load (VL) suppression is critical to protect global HIV response investments. We describe VL scale-up between 2017–2020 in the RSS President’s Emergency Plan for AIDS Relief (PEPFAR)-supported program. Methods President’s Emergency Plan for AIDS Relief (PEPFAR) South Sudan developed a VL scale-up plan and tools spanning the VL cascade: pre-test, test and post-test and included assessment of clinical facility and laboratory readiness; clinical and laboratory forms and standard operating procedures for test ordering, specimen collection, processing, results return and utilization; procedures to map clients, monitor turn-around-times (TAT), and an electronic system to monitor VL performance. Results Between 2017 to 2020, VL monitoring was established in 58 facilities, with 59,600 VL samples processed, and improvements in TAT (150–28 days) and rejection rates (1.9%–0.8%). VL documentation improved for dates of ART initiation, VL test request and dispatch, and HIV regimen. Total average time from high VL to repeat VL decreased from 15.9 months to 6.4 months in 2017 and 2019, respectively. Conclusions A concerted approach to VL scale-up has been fundamental as South Sudan strives towards UNAIDS 95-95-95 targets for PWH on ART.

HIV transmission can occur with a viral load of at least 200 copies per mL of blood and low-level viraemia can lead to virological failure; the threshold level at which risk for virological failure is conferred is uncertain. To better understand low-level viraemia prevalence and outcomes, we analysed retrospective longitudinal data from a large cohort of people living with HIV on antiretroviral therapy (ART) in Nigeria.In this retrospective cohort study using previously collected longitudinal patient data, we estimated rates of virological suppression (≤50 copies per mL), low-level viraemia (51-999 copies per mL), virological non-suppression (≥1000 copies per mL), and virological failure (≥2 consecutive virological non-suppression results) among people living with HIV aged 18 years and older who initiated and received at least 24 weeks of ART at 1005 facilities in 18 Nigerian states. We analysed risk for low-level viraemia, virological non-suppression, and virological failure using log-binomial regression and mixed-effects logistic regression.At first viral load for 402 668 patients during 2016-21, low-level viraemia was present in 64 480 (16·0%) individuals and virological non-suppression occurred in 46 051 (11·4%) individuals. Patients with low-level viraemia had increased risk of virological failure (adjusted relative risk 2·20, 95% CI 1·98-2·43; p0·0001). Compared with patients with virological suppression, patients with low-level viraemia, even at 51-199 copies per mL, had increased odds of low-level viraemia and virological non-suppression at next viral load; patients on optimised ART (ie, integrase strand transfer inhibitors) had lower odds than those on non-integrase strand transfer inhibitors for the same low-level viraemia range (eg, viral load ≥1000 copies per mL following viral load 400-999 copies per mL, integrase strand transfer inhibitor: odds ratio 1·96, 95% CI 1·79-2·13; p0·0001; non-integrase strand transfer inhibitor: 3·21, 2·90-3·55; p0·0001).Patients with low-level viraemia had increased risk of virological non-suppression and failure. Programmes should revise monitoring benchmarks and targets from less than 1000 copies per mL to less than 50 copies per mL to strengthen clinical outcomes and track progress to epidemic control.None.

International audience; Background: Global HIV-1 genetic diversity and evolution form a major challenge to treatment and prevention efforts. An increasing number of distinct HIV-1 recombinants have been identified worldwide, but their contribution to the global epidemic is unknown. We aimed to estimate the global and regional distribution of HIV-1 recombinant forms during 1990-2015.Methods: We assembled a global HIV-1 molecular epidemiology database through a systematic literature review and a global survey. We searched the PubMed, Embase (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) databases for HIV-1 subtyping studies published from Jan 1, 1990, to Dec 31, 2015. Unpublished original HIV-1 subtyping data were collected through a survey among experts in the field who were members of the WHO-UNAIDS Network for HIV Isolation and Characterisation. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. Countries were grouped into 14 regions and analyses were done for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV in each country to generate regional and global estimates of numbers and proportions of HIV-1 recombinants in each time period. The systematic review is registered with PROSPERO, CRD42017067164.Findings: Our global data collection yielded an HIV-1 molecular epidemiology database of 383 519 samples from 116 countries in 1990-2015. We found that the proportion of recombinants increased over time, both globally and in most regions, reaching 22·8% (7 978 517 of 34 921 639) of global HIV-1 infections in 2010-15. Both the proportion and the number of distinct CRFs detected increased over time to 16·7% and 57 CRFs in 2010-15. The global and regional distribution of HIV-1 recombinants was diverse and evolved over time, and we found large regional variation in the numbers (0-44 CRFs), types (58 distinct CRFs), and proportions (0-80·5%) of HIV-1 recombinants. Globally, CRF02_AG was the most prevalent recombinant, accounting for 33·9% (2 701 364 of 7 978 517) of all recombinant infections in 2010-15. URFs accounted for 26·7% (2 131 450 of 7 978 517), CRF01_AE for 23·0% (1 838 433), and other CRFs for 16·4% (1 307 270) of all recombinant infections in 2010-15. Although other CRFs accounted for small proportions of infections globally (

Increasing the volume, strengthening the quality, and proactively using data of human immunodeficiency virus (HIV) load testing are pivotal to limiting the threat of HIV drug resistance (HIVDR) accumulation, and allow for optimal case-based HIVDR surveillance. Triangulation of viral load (VL) and HIVDR testing data could be pursued to answer key questions and translate data and results for program and public policy. Identification of virologic failure and early management mitigates the greater risk of HIVDR. Routine VL monitoring and evaluation systems are necessary, and countries should consider reviewing system requirements, structural needs, and procedural and technical factors for the entire VL cascade, with special emphasis on post-test result use.

Summary Scaling up access to HIV viral load testing for individuals undergoing antiretroviral therapy in low-resource settings is a global health priority, as emphasised by research showing the benefits of suppressed viral load for the individual and the whole population. Historically, large-scale diagnostic test implementation has been slow and incomplete because of service delivery and other challenges. Building on lessons from the past, in this Personal View we propose a new framework to accelerate viral load scale-up and ensure equitable access to this essential test. The framework includes the following steps: (1) ensuring adequate financial investment in scaling up this test; (2) achieving pricing agreements and consolidating procurement to lower prices of the test; (3) strengthening functional tiered laboratory networks and systems to expand access to reliable, high-quality testing across countries; (4) strengthening national leadership, with prioritisation of laboratory services; and (5) demand creation and uptake of test results by clinicians, nurses, and patients, which will be vital in ensuring viral load tests are appropriately used to improve the quality of care. The use of dried blood spots to stabilise and ship samples from clinics to laboratories, and the use of point-of-care diagnostic tests, will also be important for ensuring access, especially in settings with reduced laboratory capacity. For countries that have just started to scale up viral load testing, lessons can be learnt from countries such as Botswana, Brazil, South Africa, and Thailand, which have already established viral load programmes. This framework might be useful for guiding the implementation of viral load with the aim of achieving the new global HIV 90-90-90 goals by 2020.

The World Health Organization (WHO) recommends viral load testing as the preferred method for monitoring the clinical response of patients with human immunodeficiency virus (HIV) infection to antiretroviral therapy (ART) (1). Viral load monitoring of patients on ART helps ensure early diagnosis and confirmation of ART failure and enables clinicians to take an appropriate course of action for patient management. When viral suppression is achieved and maintained, HIV transmission is substantially decreased, as is HIV-associated morbidity and mortality (2). CDC and other U.S. government agencies and international partners are supporting multiple countries in sub-Saharan Africa to provide viral load testing of persons with HIV who are on ART. This report examines current capacity for viral load testing based on equipment provided by manufacturers and progress with viral load monitoring of patients on ART in seven sub-Saharan countries (Côte d'Ivoire, Kenya, Malawi, Namibia, South Africa, Tanzania, and Uganda) during January 2015-June 2016. By June 2016, based on the target numbers for viral load testing set by each country, adequate equipment capacity existed in all but one country. During 2015, two countries tested85% of patients on ART (Namibia [91%] and South Africa [87%]); four countries tested25% of patients on ART. In 2015, viral suppression was80% among those patients who received a viral load test in all countries except Côte d'Ivoire. Sustained country commitment and a coordinated global effort is needed to reach the goal for viral load monitoring of all persons with HIV on ART.

HIV-1 viral load (VL) levels are used for monitoring disease progression and antiretroviral therapy outcomes in HIV-infected patients. To assess the performance of laboratories conducting HIV-1 VL testing in resource-limited settings, the U.S. Centers for Disease Control and Prevention implemented a voluntary, free-of-charge, external quality assurance program using dried tube specimens (DTSs). Between 2010 and 2012, DTS proficiency testing (PT) panels consisting of 5 specimens were distributed at ambient temperature to participants. The results from the participants ( n ≥ 6) using the same assay were grouped, analyzed, and graded as acceptable within a group mean ± 3 standard deviations. Mean proficiency scores were calculated by dividing the combined PT scores by the number of testing cycles using a linear regression model. Between 2010 and 2012, the number of participants enrolled increased from 32 in 16 countries to 114 in 44 countries. A total of 78.2% of the participants reported results using 10 different VL assays. The rates of reporting of acceptable results by the participants were 96.6% for the Abbott assay, 96.3% for the Roche Cobas assay, 94.5% for the Roche Amplicor assay, 93.0% for the Biocentric assay, and 89.3% for the NucliSens assay. The overall mean proficiency scores improved over time ( P = 0.024). DTSs are a good alternative specimen type to plasma specimens for VL PT programs, as they do not require cold chain transportation and can be used on PCR-based assays. Our data suggest that the CDC HIV-1 VL PT program using DTSs positively impacts the testing performance of the participants, which might translate into better and more accurate VL testing services for patients.

Fourth-generation HIV rapid tests (RTs) claim to detect both p24 antigen (Ag) and HIV antibodies (Ab) for early identification of acute infections, important for targeting prevention and reducing HIV transmission. In a nationally representative household survey in Swaziland, 18,172 adults, age 18 to 49 years, received home-based HIV rapid testing in 2010 and 2011. Of the 18,172 individuals, 5,822 (32.0%) were Ab positive (Ab + ) by the Determine HIV-1/2 Ab/Ab combo test, and 5,789 (99.4%) of those were confirmed to be reactive in the Uni-Gold test. Determine combo identified 12 individuals as having acute infections (Ag + /Ab negative [Ab − ]); however, none had detectable HIV-1 RNA and 8 of 12 remained HIV negative at their 6-week follow-up visit (4 were lost to follow-up). All RT-nonreactive samples were pooled and tested by nucleic acid amplification testing (NAAT) to identify acute infections. NAAT identified 13 (0.1%) of the 12,338 HIV antibody-negative specimens as HIV RNA positive, with RNA levels ranging from 300 to >10,000,000 copies/ml. However, none of them were Ag + by Determine combo. Follow-up testing of 12 of the 13 NAAT-positive individuals at 6 months demonstrated 12 seroconversions (1 individual was lost to follow-up). Therefore, the Determine combo test had a sensitivity of 0% (95% confidence interval, 0 to 28) and positive predictive value of 0% for the detection of acute infections. The ability of the 4th-generation Determine combo to detect antigen was very poor in Swaziland. Thus, the Determine combo test does not add any value to the current testing algorithm; rather, it adds additional costs and complexity to HIV diagnosis. The detection of acute HIV infections may need to rely on other testing strategies.

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0–100.0) and 100.0% (95% CI: 96.0–100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0–100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9 copies/mL at 95% CIs (p = 0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

Pediatric human immunodeficiency virus (HIV) infection remains an important public health issue in resource-limited settings. In 2015, 1.4 million children aged 50% decline. The most common challenges for access to testing for early infant diagnosis included difficulties in specimen transport, long turnaround time between specimen collection and receipt of results, and limitations in supply chain management. Further reductions in HIV mortality in children can be achieved through continued expansion and improvement of services for early infant diagnosis in PEPFAR-supported countries, including initiatives targeted to reach HIV-exposed infants, ensure access to programs for early infant diagnosis of HIV, and facilitate prompt linkage to treatment for children diagnosed with HIV infection.

Swaziland has the highest national HIV prevalence worldwide. The Swaziland HIV Incidence Measurement Survey (SHIMS) provides the first national HIV incidence estimate based on prospectively observed HIV seroconversions.A two-stage survey sampling design was used to select a nationally representative sample of men and women aged 18-49 years from 14 891 households in 575 enumeration areas in Swaziland, who underwent household-based counselling and rapid HIV testing during 2011. All individuals aged 18-49 years who resided or had slept in the household the night before and were willing to undergo home-based HIV testing, answer demographic and behavioural questions in English or siSwati, and provide written informed consent were eligible for the study. We performed rapid HIV testing and assessed sociodemographic and behavioural characteristics with use of a questionnaire at baseline and, for HIV-seronegative individuals, 6 months later. We calculated HIV incidence with Poisson regression modelling as events per person-years × 100, and we assessed covariables as predictors with Cox proportional hazards modelling. Survey weighting was applied and all models used survey sampling methods.Between Dec 10, 2010, and June 25, 2011, 11 897 HIV-seronegative adults were enrolled in SHIMS and 11 232 (94%) were re-tested. Of these, 145 HIV seroconversions were observed, resulting in a weighted HIV incidence of 2·4% (95% CI 2·1-2·8). Incidence was nearly twice as high in women (3·1%; 95% CI 2·6-3·7) as in men (1·7%; 1·3-2·1, p0·0001). Among men, partner's HIV-positive status (adjusted hazard ratio [aHR] 2·67, 1·06-6·82, p=0·040) or unknown serostatus (aHR 4·64, 2·32-9·27, p0·0001) in the past 6 months predicted HIV seroconversion. Among women, significant predictors included not being married (aHR 2·90, 1·44-5·84, p=0·0030), having a spouse who lives elsewhere (aHR 2·66, 1·29-5·45, p=0·0078), and having a partner in the past 6 months with unknown HIV status (aHR 2·87, 1·44-5·84, p=0·0030).Swaziland has the highest national HIV incidence in the world. In high-prevalence countries, population-based incidence measures and programmes that further expand HIV testing and support disclosure of HIV status are needed.President's Emergency Plan for AIDS Relief (PEPFAR) by the Centers for Disease Control and Prevention.

Stakeholders agree that supporting high-quality diagnostics is essential if we are to continue to make strides in the fight against human immunodeficiency virus (HIV) and tuberculosis. Despite the need to strengthen existing laboratory infrastructure, which includes expanding and developing new laboratories, there are clear diagnostic needs where conventional laboratory support is insufficient. Regarding HIV, rapid point-of-care (POC) testing for initial HIV diagnosis has been successful, but several needs remain. For tuberculosis, several new diagnostic tests have recently been endorsed by the World Health Organization, but a POC test remains elusive. Human immunodeficiency virus and tuberculosis are coendemic in many high prevalence locations, making parallel diagnosis of these conditions an important consideration. Despite its clear advantages, POC testing has important limitations, and laboratory-based testing will continue to be an important component of future diagnostic networks. Ideally, a strategic deployment plan should be used to define where and how POC technologies can be most efficiently and cost effectively integrated into diagnostic algorithms and existing test networks prior to widespread scale-up. In this fashion, the global community can best harness the tremendous capacity of novel diagnostics in fighting these 2 scourges.

Background The best current animal model for HIV infection and evaluation of antiviral compounds is the Simian–human immunodeficiency virus (SHIV)/macaque system. There are multiple recombinant SHIVs available, but these viruses have limitations in evaluating combination drug strategies for prevention. Drug combinations that target reverse transcriptase (RT, either nRTI or nnRTI) and envelope (entry or fusion inhibitors) have to be tested separately, which does not permit the assessment of additive, synergistic, or antagonistic effects of ARV combinations. We describe construction of a dual SHIV containing both HIV RT and a CCR5-specific HIV envelope gene in a simian immunodeficiency virus backbone. Methods The RT Env SHIV molecular clone was constructed using RT SHIV and SHIV162p3 sequences as templates to generate RT Env SHIV. RT Env SHIV was expanded in vitro in CD8-depleted macaque peripheral blood mononuclear cells (PBMC). Recombinant virus was used to infect a rhesus macaque (4.3 × 104 tissue culture infectious dose [TCID50], intravenously [IV]). A second passage in a macaque by IV transfer of 10 ml of blood obtained from the first infection was also done. The in vivo adapted virus stock from these macaques was used to produce high titer stocks in vitro and used to rectally infect an additional macaque. Results Peak viral load reached 6 × 105 vRNA copies/ml in plasma in both IV-exposed macaques and remained detectable in the one animal for 16 weeks after infection. A viral stock (1.68 × 104 TCID50) derived from the second macaque passage has been produced in CD8-depleted rhesus PBMC and was successfully used to demonstrate mucosal transmission. The resulting RT Env SHIV retained the sensitivity to HIV RT and entry inhibitors of its parental viruses. Conclusions The objective of this study was to develop and characterize a SHIV recombinant virus for evaluating the efficacy of ART and microbicide products that target both HIV RT and/or Env-mediated entry. RT Env SHIV can productively infect macaques by both the IV and mucosal route, making it a valuable tool for transmission studies.

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex antigenically. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

To achieve global targets for universal treatment set forth by the Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) (UNAIDS), viral load monitoring for HIV-infected persons receiving antiretroviral therapy (ART) must become the standard of care in low- and middle-income countries (LMIC) (1). CDC and other U.S. government agencies, as part of the President's Emergency Plan for AIDS Relief, are supporting multiple countries in sub-Saharan Africa to change from the use of CD4 cell counts for monitoring of clinical response to ART to the use of viral load monitoring, which is the standard of care in developed countries. Viral load monitoring is the preferred method for immunologic monitoring because it enables earlier and more accurate detection of treatment failure before immunologic decline. This report highlights the initial successes and challenges of viral load monitoring in seven countries that have chosen to scale up viral load testing as a national monitoring strategy for patients on ART in response to World Health Organization (WHO) recommendations. Countries initiating viral load scale-up in 2014 observed increases in coverage after scale-up, and countries initiating in 2015 are anticipating similar trends. However, in six of the seven countries, viral load testing coverage in 2015 remained below target levels. Inefficient specimen transport, need for training, delays in procurement and distribution, and limited financial resources to support scale-up hindered progress. Country commitment and effective partnerships are essential to address the financial, operational, technical, and policy challenges of the rising demand for viral load monitoring.

An accurate accessible test for early infant diagnosis (EID) is crucial for identifying HIV-infected infants and linking them to treatment. To improve EID services in Ukraine, dried blood spot (DBS) samples obtained from 237 HIV-exposed children (≤18 months of age) in six regions in Ukraine in 2012 to 2013 were tested with the AmpliSens DNA-HIV-FRT assay, the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 Qual test, and the Abbott RealTime HIV-1 Qualitative assay. In comparison with the paired whole-blood results generated from AmpliSens testing at the oblast HIV reference laboratories in Ukraine, the sensitivity was 0.99 (95% confidence interval [CI], 0.95 to 1.00) for the AmpliSens and Roche CAP/CTM Qual assays and 0.96 (95% CI, 0.90 to 0.98) for the Abbott Qualitative assay. The specificity was 1.00 (95% CI, 0.97 to 1.00) for the AmpliSens and Abbott Qualitative assays and 0.99 (95% CI, 0.96 to 1.00) for the Roche CAP/CTM Qual assay. McNemar analysis indicated that the proportions of positive results for the tests were not significantly different ( P > 0.05). Cohen's kappa (0.97 to 0.99) indicated almost perfect agreement among the three tests. These results indicated that the AmpliSens DBS and whole-blood tests performed equally well and were comparable to the two commercially available EID tests. More importantly, the performance characteristics of the AmpliSens DBS test meets the World Health Organization EID test requirements; implementing AmpliSens DBS testing might improve EID services in resource-limited settings.

Characterization of human immunodeficiency virus (HIV)-1-specific immune responses against subtypes circulating in areas where the virus is endemic is critical for the design of candidate vaccines. In Cote d'Ivoire, the most prevalent HIV-1 subtype is CRF02_AG. We detected T cell responses to CRF02_AG consensus p24(gag) or protease peptides in 81% of HIV-1- or HIV-1/2-infected blood donors in Abidjan, Cote d'Ivoire. Both the magnitude and the breadth of interferon- gamma enzyme-linked immunospot responses were inversely correlated with plasma viral load. One frequently recognized peptide in p24(gag) was mapped to the optimal epitope (TPQDLNMML). Further studies of this epitope may be important for the development of HIV-1 vaccines for West Africa and West-Central Africa.

We developed an AIDS vaccine for Western and West-Central Africa founded on HIV-1 subtype CRF02_AG. Rhesus macaques were primed with Gag–Pol–Env-expressing plasmid DNA and boosted with a recombinant modified vaccinia virus Ankara (rMVA), expressing matched proteins. Two DNA vaccine constructs (IC1–90 and IC48) that differed by point mutations in gag and pol were compared. IC1–90 produces primarily immature (core comprises unprocessed Pr55Gag) HIV-like particles (VLPs) and IC48 produces mature VLP with processed Pr55Gag, immature VLP, and intracellular protein aggregates. Both vaccines raised significant cellular responses for Gag, Pol, and Env. Approximate twofold higher ELISPOT responses to Gag and Env epitopes were observed for IC48 animals than for IC1–90 animals at the peak post-MVA effector (P = 0.028) and late memory (P = 0.051) phases, respectively. Greater breadth for IC48-primed animals was observed than for IC1–90-primed animals at peak response (P = 0.03). Our results indicated that the vaccines elicited high frequency T cell responses and primed anti-Env antibody. They also suggest that expression of different forms of VLP has a significant effect on elicited cellular and humoral immunity.

One of the unknowns faced by an HIV/AIDS vaccine is the ability of a single clade vaccine to protect against the multiple genetic subtypes and recombinant forms of HIV-1 present in the current pandemic. Here, we use a macaque model to investigate the ability of our clade B vaccine that consists of DNA priming and modified vaccinia Ankara (MVA) virus boosting to elicit T cell responses that recognize an A/G recombinant of HIV- 1. To test for cross-reactive T cells, intracellular cytokine staining was conducted using five pools of Gag and six pools of Env peptides representing B or A/G sequences. Studies using the peptide pools revealed essentially complete conservation of the CD8 response but only ∼50% conservation of the CD4 response. Thus, the ability of an HIV vaccine for one clade to protect against other clades may be more limited by the ability to provide CD4 T cell help than the ability to elicit CD8 effector functions.

Recently, a vaccine consisting of DNA priming followed by boosting with modified vaccinia Ankara (MVA) has provided long-term protection of rhesus macaques against a virulent challenge with a chimera of simian and human immunodeficiency viruses. Here, we report studies on the development of the DNA component for a DNA/MVA HIV vaccine for humans. Specifically, we assess the ability of a codon-optimized Gag-expressing DNA and two noncodon-optimized Gag-Pol-Env-expressing DNAs to prime the MVA booster dose. The codon-optimized DNA expressed virus-like particles (VLPs), whereas one of the noncodon-optimized DNAs expressed VLPs and the other expressed aggregates of HIV proteins. The MVA boost expressed Gag-Pol and Env and produced VLPs. Immunogenicity studies in macaques used one intramuscular prime with 600 microg of DNA and two intramuscular boosts with 1 x 10(8) pfu of MVA at weeks 8 and 30. The codon-optimized and noncodon-optimized DNAs proved similar in their ability to prime anti-Gag T cell responses. The aggregate and VLP-expressing Gag-Pol-Env DNAs also showed no significant differences in their ability to prime anti-Env Ab responses. The second MVA booster dose did not increase the peak CD4 and CD8 T cell responses, but increased anti-Env Ab titers by 40- to 90-fold. MVA-only immunizations elicited 10-100 times lower frequencies of T cells and 2-4 lower titers of anti-Env Ab than the Gag-Pol-Env DNA/MVA immunizations. Based on the breadth of the T cell response and a trend toward higher titers of anti-Env Ab, we are moving forward with human trials of the noncodon-optimized VLP-expressing DNA.

Recently, a simian/human immunodeficiency virus (SHIV) vaccine consisting of priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia Ankara (rMVA) has successfully controlled a virulent SHIV challenge in a macaque model. In this, and the accompanying paper, we report on the construction and testing of a Gag-Pol-Env DNA/MVA vaccine for HIV-1/AIDS. The DNA vaccine, pGA2/JS2, expresses aggregates of Gag proteins and includes safety mutations that render it integration, reverse transcription, and packaging defective. The rMVA vaccine, MVA/HIV 48, is integration and reverse transcription defective and has a truncated Env to enhance expression on the plasma membrane. In a study in rhesus macaques, priming with pGA2/JS2 and boosting with MVA/HIV 48 raised high frequencies of T cells for Gag and Env and lower frequencies of T cells for PR, RT, and Tat. Stimulations with five peptide pools for Gag and seven peptide pools for Env revealed epitopes for cellular immune responses throughout Gag and Env. On average, CD4 T cells from the vaccinated animals recognized 7.1 peptide pools and CD8 T cells, 3.2 peptide pools. Both the height and the breadth of the elicited cellular response provide hope that this multiprotein DNA/MVA vaccine will successfully control clade B isolates of HIV-1, as well as contribute to the control of other clades and recombinant forms of HIV-1/AIDS.

We developed an AIDS vaccine for Western and West-Central Africa based on a DNA plasmid vector expressing HIV-1 recombinant subtype CRF02_AG gag , pol , and env genes. To optimize the production of noninfectious HIV-like particles (VLPs) and potentially improve the effectiveness of the vaccine, we generated four potential vaccine constructs: the parental (IC2) and three modifications (IC25, IC48, and IC90) containing mutations within the HIV protease. While the parental construct IC2 expressed aggregates of Gag proteins, the IC25 construct resulted in the production of immature VLPs (the core comprises unprocessed Pr 55Gag ). The remaining two constructs (IC48 and IC90) produced mature VLPs (the core comprises processed capsid p24) in addition to immature VLPs and aggregates of Gag proteins. VLPs incorporated significant levels of mature gp120 envelope glycoprotein. Importantly, the mature VLPs were fusion competent and entered coreceptor-specific target cells. The production of multiple antigenic forms, including fusion-competent VLPs, by candidate DNA vaccine constructs may provide immunologic advantages for induction of protective cellular and humoral responses against HIV-1 proteins.

We performed a comparative analysis between Roche Amplicor HIV-1 DNA test and CAPTAQ assay for the detection of HIV in 830 dried blood spot (DBS) pediatric samples collected in Mozambique. Our results demonstrated no statistical difference between these assays. The CAPTAQ assay approached nearly 100% repeatability/accuracy. The increased throughput of testing with minimal operator interference in performing the CAPTAQ assay clearly demonstrated that this method is an improvement over the Roche Amplicor HIV-1 DNA test, version 1.5.

We considered several key issues regarding the development of a DNA-based human immunodeficiency virus type 1 (HIV-1) vaccine: (1) should the candidate vaccine construct be derived from incident or prevalent HIV-1 strains; and (2) should circulating plasma virus, archived HIV-1 provirus recovered from peripheral blood mononuclear cells, or both be included? To address these questions, we collected circulating HIV-1 strains from infected individuals residing in Abidjan, Côte d'Ivoire. From a panel of 23 strains, 22 were HIV-1 subtype A in gag, 19 of which phylogenetically clustered with the recombinant HIV-1, CRF02-AG strains from West Africa. The mosaic genome of CRF02-AG was confirmed by sequencing the protease gene. A consensus gag p24 protein sequence was generated and 147 of 148 codons were identical to CRF02-AG (IbNG). Regardless of the sequence origin (RNA, provirus, incident, or prevalent), the gag p24 consensus sequences were highly representative of these distinct virologic compartments. These data suggest that the consensus sequence generated from incident and prevalent infections may provide an appropriate sequence for a DNA vaccine and is largely representative of the major circulating viral strain.

Background Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Methods Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Results Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2-99.2] and 98.1% [95%CI: 89.7-100]) and those for DBS (93.9% [95%CI: 88.8-97.2] and 88.0% [95%CI: 82.2-92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4-97.7) and 73.6 (95%CI: 51.6-86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6-100.0] vs. 94.7% [95%CI: 89.8-97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4-13.7] compared to DPS: 94.0%, [95% CI: 83.5-98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and specificity of 96.6% [95% CI 91.8-98.7] and 90.4% [95% CI 78.2-96.4] respectively. The optimum threshold was at 3000 cps/ml with an accuracy of 95.5%, sensitivity and specificity of 94.6% [95%CI 89.3-97.5] and 98.1% [95%CI 88.4-99.9]) respectively. The best threshold for CAP/CTM was at 4000 copies /mL, with 92.5% accuracy (sensitivity of 96.0% [95%CI 91.0-98.3] and specificity of 82.7% [95%CI 69.2-91.3]). Conclusions There was similar performance between matched DBS, DPS and plasma using the Abbott test, and good correlation for matched DPS and plasma using the CAPCTM test. The findings suggest that DBS and DPS may be reliably used as alternative specimens to plasma to measure HIV-1 VL using Abbott, and DPS may be reliably used with CAP/CTM in resource-limited settings.

The 2013 WHO antiretroviral therapy (ART) guidelines recommend dried blood spots (DBS) as an alternative specimen type for viral load (VL) monitoring. We assessed the programmatic utility of screening for antiretroviral (ARV) treatment failure (TF) at 5,000 and 1,000 copies/ml using DBS and dried plasma spots (DPS) with a commonly used VL assay, the Roche Cobas Ampliprep/Cobas TaqMan V.2.0 (CAP/CTM). Plasma, DBS, and DPS were prepared from 839 whole-blood specimens collected from patients on ART for ≥6 months at three public facilities in Namibia. Using the CAP/CTM test, VL were measured in plasma, DBS, and DPS, and the results were compared using the plasma VL as the reference standard. The clinical sensitivities, specificities, and positive (PPV) and negative predictive values (NPV) of DBS at ARV TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml were 0.99, 0.55, 0.33, and 0.99 and 0.99, 0.26, 0.29, and 0.99, respectively, and for DPS at TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml, they were 0.88, 0.98, 0.92, and 0.97 and 0.91, 0.96, 0.89, and 0.97, respectively. The prevalences of TF were overestimated in DBS by 33% and 57% at these two thresholds, respectively. A high rate of false-positive results would occur if the CAP/CTM with DBS were to be used to screen for ARV TF. WHO recommendations for DBS-based VL monitoring should be specific to the VL assay version and type. Despite the better performance of DPS, the programmatic utility for TF screening may be limited by requirements for processing the whole blood at the collection site.

A voluntary, cost-free external quality assessment (EQA) program established by the U.S. Centers for Disease Control and Prevention (CDC) was implemented to primarily monitor the performance of laboratories conducting HIV Early Infant Diagnosis (EID) from dried blood spots (DBS) in low- to middle-income countries since 2006. Ten blind DBS proficiency test (PT) specimens and 100 known HIV-positive and -negative DBS specimens (to be used as internal controls) were shipped triannually to participating laboratories with reports for the PT specimens due within 30 days. The participant's results and a summary of the performance of all participating laboratories and each diagnostic method were provided after each test cycle. Enrollment in the CDC PT program expanded progressively from 17 laboratories from 11 countries in 2006 to include 136 laboratories from 41 countries at the end of 2012. Despite external pressures to test and treat more children while expanding EID programs, mean PT test scores significantly improved over time as demonstrated by the upward trend from mid-2006 to the end of 2012 ( P = 0.001) and the increase in the percentage of laboratories scoring 100% ( P = 0.003). The mean test scores plateaued over the past 10 testing cycles, ranging between 98.2% and 99.7%, and discordant test results still occur but at a rate of no higher than 2.6%. Analysis of these test results suggests a positive impact of proficiency testing on the testing performance of the participating laboratories, and a continuous training program and proficiency testing participation may translate into laboratories improving their testing accuracy.

A population-based surveillance registry was used to identify human immunodeficiency virus (HIV)-infected persons in the United States at increased risk for group O and group N infections (those born in or near African countries where group O infection has been reported). Of 155 eligible subjects, 37 gave samples. By phylogenetic and serologic analysis, 32 were infected with group M (16 with subtype A, 5 with B, 7 with C, and 1 each with subtypes D, F2, G, and recombinant A/J) and 2 with group O but none with group N virus. For 3, samples could not be typed by serology or amplified by polymerase chain reaction using group M-, O-, or N-specific primers. In the United States, group O HIV infection is uncommon; no case of group N infection was found. African-born persons may have HIV strains typical of their birth country. Ongoing subtype surveillance may allow early identification of novel or emerging HIV strains.

Persons who were human immunodeficiency virus type 1 (HIV-1)-infected but who remained persistently seronegative (HIPS) on HIV-1 antibody tests were examined through AIDS case surveillance. Six such individuals (HIPS-1 to -4, -7, and -9) were examined to determine whether their persistent seronegativity was attributable to immune dysfunction or infection with atypical HIV. Of the 6, 4 had antibody titers to at least 1 other common pathogen. In vitro stimulation of peripheral blood mononuclear cells from HIPS-4 and HIPS-7 with pokeweed mitogen or phosphorothioate oligodeoxynucleotide (direct B cell mitogen) did not produce HIV-1-specific antibody. Reconstitution experiments with recombinant interleukin (rIL)-4 and rIL-12 also had no impact on antibody production. Virus isolates from HIPS-4 and -9 were R5X4-tropic, whereas HIPS-7 was CCR5-tropic only. Sequence analysis of long terminal repeat, p24, and env gp41 did not reveal any specific mutation, and phylogenetic analysis confirmed that all 6 virus specimens were HIV-1 subtype B. These data suggest that the lack of a detectable antibody response in these patients may be the result of immune dysfunction.

The potential to establish dual retroviral infections was investigated in this study. Groups of macaques infected with human immunodeficiency virus type 2 (HIV-2) isolate (either GB122 or CDC77618) were exposed to the other virus at 2, 4, 8, 12, 14, or 72 weeks after primary inoculation. Dual infections were established in macaques simultaneously exposed to both viruses. In other groups, secondary infections were observed only if challenge occurred at early intervals after primary infection but before a full seroconversion. Polymerase chain reaction and virus-isolation data demonstrated that challenges at 8, 12, 14, or 72 weeks after infection with the initial isolate failed to result in a dual infection. Anti-HIV-2 serologic titers, CD4 levels, virus burden, and the ability to superinfect peripheral blood mononuclear cells in vitro were not correlated with susceptibility to or protection from secondary challenges in this investigation. These findings demonstrate a window period for susceptibility to dual infection and indicate that protection from retroviral infection may be achievable.

We analyzed the genetic variability and phylogenetic relationships among 28 HIV-2 strains collected from patients enrolled in an HIV epidemiologic study in Abidjan, Ivory Coast, during 1995-1996. Although both subtype A (n = 8; 29%) and subtype B (n = 20; 71%) were present in this sampling, the majority of infections were caused by subtype B viruses. These findings contrasted with the reported predominance of HIV-2 subtype A in other African countries. The broad genetic diversity identified among protease gene sequences for HIV-2 subtype A (6%; range 3-15%) and subtype B (7%; range, 2-12%), and their presence in Abidjan during the 1980s, document a long coexistence of two viral subtypes in Ivory Coast. Our data indicate that viruses of subtypes A and B have contributed to the HIV-2 epidemic in Ivory Coast.

OBJECTIVE: To describe persons with HIV infection and AIDS but with persistently negative HIV antibody enzyme immunoassay (EIA) results. DESIGN: Surveillance for persons meeting a case definition for HIV-1-seronegative AIDS. SETTING: United States and Canada. PATIENTS: A total of eight patients with seronegative AIDS identified from July 1995 through September 1997. MAIN OUTCOME MEASURES: Clinical history of HIV disease, history of HIV test results, and CD4 cell counts from medical record review; results of testing with a panel of EIA for antibodies to HIV-1, and HIV-1 p24 antigen; and viral subtype. RESULTS: Negative HIV EIA results occurred at CD4 cell counts of 0-230 x 10(6)/l, and at HIV RNA concentrations of 105,000-7,943,000 copies/ml. Using a panel of HIV EIA on sera from three patients, none of the HIV EIA detected infection with HIV-1, and signal-to-cut-off ratios were < or = 0.8 or all test kits evaluated. Sera from five patients showed weak reactivity in some HIV EIA, but were non-reactive in other HIV EIA. All patients were infected with HIV-1 subtype B. CONCLUSIONS: Rarely, results of EIA tests for antibodies to HIV-1 may be persistently negative in some HIV-1 subtype B-infected persons with AIDS. Physicians treating patients with illnesses or CD4 cell counts suggestive of HIV infection, but for whom results of HIV EIA are negative, should consider p24 antigen, nucleic acid amplification, or viral culture testing to document the presence of HIV.

Infections following repeated, low-dose (RLD), mucal S(H)IV exposures of macaques are used to model sexual HIV exposures for biomedical prevention testing. Different susceptibilities among animals can complicate study designs. In rhesus macaques, TRIM5 alleles Q, CypA, and TFP are resistance factors for infection with some S(H)IV strains, but not for SIVmac239 due to its capsid properties. SIVmac239-derived SHIVSF162P3 has been demonstrated to reproducibly infect mucosally in vaginal and rectal RLD models. To further test the suitability of SHIVSF162P3 for RLD models, we studied the influence of the TRIM5 genotype on susceptibility to rectal RLD infection and on plasma viremia by analyzing 43 male Indian rhesus macaques from control arms of completed studies. The median number of exposures required for infection was three (Q/Q, n=4) (TRIM5 alleles, number of macaques, respectively), four (Q/CypA, n=7), three (TFP/Q, n=15), three (TFP/TFP, n=15), and two (TFP/CypA, n=2); TRIM5(CypA/CypA) was not represented in our study. Median peak viremia (log10 viral copies/ml) in infected animals was 7.4 (Q/Q, n=4), 7.2 (Q/CypA, n=6), 7.3 (TFP/Q, n=13), 7.1 (TFP/TFP, n=15), and 6.5 (TFP/CypA; n=2). Neither susceptibility nor peak viremia was significantly different (log rank test, Kruskal-Wallis test, respectively). Rhesus macaques' susceptibility to RLD SHIVSF162P3 is independent of the TRIM5 TFP, CypA, and Q alleles, with the limitation that the power to detect any impact of CypA/CypA and TFP/CypA genotypes was nonexistent or low, due to absence or infrequency, respectively. The finding that TRIM5 alleles do not restrict mucosal infection or ensuing replication rates suggests that SHIVSF162P3 is indeed suitable for RLD experimentation.

Participation in external quality assessment programs is critical to ensure quality clinical laboratory testing. Commercially available proficiency test panels for HIV-1 virus load testing that are used commonly in external quality assessment programs remain a financial obstacle to resource-limited countries. Maintaining cold-chain transportation largely contributes to the cost of traditional liquid proficiency test panels. Therefore, we developed and evaluated a proficiency test panel using dried tube specimens that can be shipped and stored at ambient temperature. This dried tube specimens panel consisted of 20 μl aliquots of a HIV-1 stock that were added to 2 ml tubes and left uncapped for drying, as a preservation method. The stability of dried tube specimens at concentrations ranging from 10² to 10⁶·⁵ RNA copies/ml was tested at different temperatures over time, showing no viral load reduction at 37 °C and a decrease in viral load smaller than 0.5 Log₁₀ at 45 °C for up to eight weeks when compared to initial results. Eight cycles of freezing-thawing had no effect on the stability of the dried tube specimens. Comparable viral load results were observed when dried tube specimen panels were tested on Roche CAPTAQ, Abbott m2000, and Biomerieux easyMAG viral load systems. Preliminary test results of dried proficiency test panels shipped to four African countries at ambient temperature demonstrated a low inter assay variation (SD range: 0.29-0.41 Log₁₀ RNA copies/ml). These results indicated that HIV-1 proficiency test panels generated by this methodology might be an acceptable alternative for laboratories in resource-limited countries to participate in external quality assessment programs.

Background The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4–6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. Objectives The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. Study design DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37 °C with high humidity, and −20 °C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37 °C), at weeks 4, 8, and 18 (−20 °C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at −20 °C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. Results HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. Conclusions Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries.

More than 90% of the 370,000 pediatric human immunodeficiency virus type 1 (HIV-1) infections globally in 2009 were acquired through mother-to-child transmission (MTCT) [1], and most of these transmissions occurred in sub-Saharan Africa. MTCT of HIV-1 occurs either during late pregnancy, the intrapartum period, or breastfeeding [2, 3]. With the application of prophylactic antiretroviral (ARV) therapy and breastfeeding avoidance, MTCT is now observed in only 1–2% of at-risk infants in developed countries [4, 5]. The majority of pregnant women residing in high HIV-burden, resource-limited countries (RLCs) are still not aware of their infection status and do not receive timely intervention measures to prevent vertical transmission [6–11]. Untreated infected infants have high HIV-related morbidity and mortality. Approximately 33% of the untreated infected infants in RLCs die during their first year of life, and >50% die within their first 2 years [12]. Treating infants early greatly reduces mortality and morbidity [13]. Recognition of the importance of reducing infant HIV mortality has facilitated the development of methods to bring appropriate testing closer to pregnant and lactating mothers, to identify HIV-infected infants earlier, and to provide timely access to life-saving ARV treatment and care. New and accurate diagnostic methods have emerged in the last few years, and many of these methods have been field-validated. This diagnostic service should not comprise a stand-alone program but must be integrated into the overall mother and child health programs to achieve the goal of prevention of mother-to-child transmission (PMTCT) [14, 15]. In this chapter, we review currently available diagnostic methodologies, including their advantages and disadvantages, their testing algorithms, and their quality assurance requirements, with a particular focus on early HIV diagnosis in the breastfed infant. Further, we discuss efforts toward the development of simple, accurate, and rapid diagnostic applications.

Ribosomal protein S13 of the nematode Brugia pahangi is recognized by B and T cells from parasite-infected animals. To identify helper T cell sites on the protein, 15 overlapping synthetic peptides spanning the entire molecule (Bp17.4) were tested for their ability to stimulate lymph node and spleen cells of peptide-immunized and recombinant antigen-immunized jirds. Lymph node cells from animals immunized with peptides 6, 8, 9, 13, and 14, corresponding to Bp17.4 amino acids (AA) 50–70, 70–90, 80–100, 120–140, and 130–150, respectively, showed strong and specific responses to the homologous peptide, while only those lymph node cells from jirds immunized with peptides 8, 9, 13, and 14 proliferated in response to Bp17.4. These results suggest the existence of at least two T cell epitopes. Lymph node cells from infected jirds also responded to these peptides and to Bp17.4 (80,000 cpm). In contrast to the lymph node cells, spleen cells from microfilariapositive animals failed to mount significant responses to any of the peptides or to Bp17.4. Splenic T cell responsiveness was restored upon removal of nylon wool adherent cells, suggesting active regulation of Bp17.4 reactivity. In liquid-phase competitive inhibition immunoassays, peptides 1 (AA 1–30) and 6 (50–70) blocked antibody binding and, therefore, these regions contain conformational antibody-binding sites. This model system should prove useful for analyzing regulation of epitope-specific responses in experimental filariasis.

The macaque model of repeated SHIV exposures is increasingly used as a preclinical tool to evaluate biomedical HIV intervention strategies. It is unclear whether multiple virus exposures induce immune responses in macaques, as documented in uninfected individuals repeatedly exposed to HIV. We here address whether repeated, rectal SHIV(SF162P3) exposures lead to systemic T cell activation in 12 rhesus macaques, and whether this is associated with increased infection resistance. Eight macaques became systemically infected after 2-7 exposures, three macaques were less susceptible (infection after 10-12 exposures), and one macaque remained uninfected after 14 exposures. PBMCs were retrospectively monitored for increases in T cell activation by analyzing the proportion of CD8(+) T cells, recently activated or proliferated T cells (markers CD38, Ki67), a marker for cytotoxicity (granzyme B), or T cell-produced plasma cytokines (IFN-gamma, RANTES, IL-2). Repeated virus exposures did not induce sustained, potent, or diverse T cell responses prior to systemic infection. Some changes occurred in the analyzed parameters during repeated virus exposures, but similar T cell activities were also observed in five SHIV-unexposed control macaques. Thus, we found no evidence that delayed infection or resistance to infection was associated with systemic, long-lasting, protective T cell responses to repeated rectal virus exposures. Our results provide further insights into the repeat exposure macaque model. We find that this model can be used for testing biomedical prevention strategies without concern of eliciting a systemic vaccination effect.

We reported previously the absence of systemic infection in a subset of macaques vaccinated with an HIV-1 DNA/MVA vaccine after 18 or more rectal challenges with low (physiologically relevant) doses of SHIV162P3. To further study the apparent protection, we looked for sequestered virus in gut tissues, lymph nodes, spleen, and testes obtained at necropsy using virus co-culture and nested PCR for SIV Gag, Pol and LTR. There was no evidence of sequestered virus in tissues obtained from the four protected macaques. In contrast, at least one tissue from each of 11 infected animals scored positive by one of these sensitive procedures. Activated PBMC from the protected macaques were not inherently resistant to in vitro infection by the challenge virus. These findings demonstrate that some vaccinated macaques appeared to be free from the challenge virus. Therefore, such T cell-based vaccines may provide some protection when challenge virus doses approach physiological equivalencies.

Historically, HIV vaccines specifically designed to raise cellular immunity resulted in protection from disease progression but not infection when tested in monkeys challenged with a single high virus exposure. An alternative approach, more analogous to human sexual exposures, is to repetitively challenge immunized monkeys with a much lower dose of virus until systemic infection is documented. Using these conditions to mimic human sexual transmission, we found that a multi-protein DNA/MVA HIV-1 vaccine is indeed capable of protecting rhesus monkeys against systemic infection when repeatedly challenged with a highly heterologous immunodeficiency virus (SHIV). Furthermore, this repetitive challenge approach allowed us to calculate per-exposure probability of infection, an observed vaccine efficacy of 64%, and undertake a systematic analysis for correlates of protection based on exposures needed to achieve infection. Therefore, improved non-human primate models for vaccine efficacy can provide novel insight and perhaps renew expectations for positive outcomes of human HIV clinical trials.

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.