FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition.

After over three decades of research, an effective anti-HIV vaccine remains elusive. Unconventional and novel vaccine strategies are needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provides greater than 80% protection of Mauritian cynomolgus macaques (MCMs) against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses are correlated with vaccine efficacy. The PCS vaccine does not induce immune activation and inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune environment generated by the PCS vaccine predicts vaccine efficacy. Our study demonstrates for the first time that a novel vaccine which targets viral maturation, but lacks full Env and Gag proteins as immunogens, can prevent intravaginal infection in a highly stringent NHP/SIV challenge model. Targeting HIV maturation thus offers a novel approach to developing an effective HIV vaccine. One Sentence Summary The anti-PCS T cell responses and the immune environment induced by the novel PCS vaccine are key correlates of vaccine efficacy

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline (P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 (P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1β (P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α (P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels (P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines.

TILRR (Toll-like interleukin-1 receptor regulator), a transcript variant of FREM1, is a novel regulatory component, which stimulates innate immune responses through binding to IL-1R1 (Interleukin-1 receptor, type 1) and TLR (Toll-like receptor) complex. However, it is not known whether TILRR expression influences other genes in the NFκB signal transduction and pro-inflammatory responses. Our previous study identified FREM1 as a novel candidate gene in HIV-1 resistance/susceptibility in the Pumwani Sex worker cohort. In this study, we investigated the effect of TILRR overexpression on expression of genes in the NFκB signaling pathway in vitro. The effect of TILRR on mRNA expression of 84 genes related to NFκB signal transduction pathway was investigated by qRT-PCR. Overexpression of TILRR on pro-inflammatory cytokine/chemokine(s) secretion in cell culture supernatants was analyzed using Bioplex multiplex bead assay. We found that TILRR overexpression significantly influenced expression of many genes in HeLa and VK2/E6E7 cells. Several cytokine/chemokine(s), including IL-6, IL-8 (CXCL8), IP-10, MCP-1, MIP-1β, and RANTES (CCL5) were significantly increased in the cell culture supernatants following TILRR overexpression. Although how TILRR influences the expression of these genes needs to be further studied, we are the first to show the influence of TILRR on many genes in the NFκB inflammatory pathways. The NFκB inflammatory response pathways are extremely important in microbial infection and pathogenesis, including HIV-1 transmission. Further study of the role of TILRR may identify the novel intervention targets and strategies against HIV infection.

The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model's true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.

The development of an effective HIV vaccine continues to be a major health challenge since, so far, only the RV144 trial has demonstrated a modest clinical efficacy. Recently, the targeting of the 12 highly conserved protease cleavage sites (PCS1–12) has been presented as a strategy seeking to hamper the maturation and infectivity of HIV. To pursue this line of research, and because peptide antigens have low immunogenicity, we have included these peptides in engineered nanoparticles, aiming at overcoming this limitation. More specifically, we investigated whether the covalent attachment of a PCS peptide (PCS5) to polysaccharide-based nanoparticles, and their coadministration with polyinosinic:polycytidylic acid (poly(I:C)), improved the generated immune response. To this end, PCS5 was first conjugated to two different polysaccharides (chitosan and hyaluronic acid) through either a stable or a cleavable bond and then associated with an oppositely charged polymer (dextran sulfate and chitosan) and poly(I:C) to form the nanoparticles. Nanoparticles associating PCS5 by ionic interactions were used in this study as the control formulation. In vivo, all nanosystems elicited high anti-PCS5 antibodies. Nanoparticles containing PCS5 conjugated and poly(I:C) seemed to induce the strongest activation of antigen-presenting cells. Interestingly, T cell activation presented different kinetics depending on the prototype. These findings show that both the nanoparticle composition and the conjugation of the HIV peptide antigen may play an important role in the generation of humoral and cellular responses.

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p<0.0001). The effect of the mucosal antibody responses in protection from repeated low dose pathogenic SIVmac251 challenges is being evaluated.

Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCMs) is an increasingly important nonhuman primate model for HIV vaccine research.We previously reported that in MCMs anti-SIV antibodies can be naturally developed without exogenous infection or vaccination, and that a vaccine targeting SIV protease cleavage sites (PCS) can cross-induce antibodies to non-PCS SIV antigens.We speculate that this is potentially caused by the existence of endogenous SIV-like antigens.External stimuli (such as environmental factors and vaccination) may induce expression of endogenous SIV-like antigens to elicit these antibodies.Database and mass spectrometry analyses were conducted to search for such antigens.We identified endogenous SIV-like DNA sequences in cynomolgus macaque genome and non-PCS peptide homologous to SIV Env protein in PBMCs of a PCS-vaccinated monkey.Our preliminary insights suggest that endogenous SIV-like antigens may be one of the possible reasons for the natural and cross-inducible SIV antibodies in MCMs.

HIV preferentially infects activated CD4+ T cells and mutates rapidly. The classical vaccine approach aimed to generate broad immune responses to full HIV proteins largely failed to address the potential adverse impact of increased number of activated CD4+ T cells as viral targets. Learning from natural immunity observed in a group of HIV resistant Kenyan female sex workers, we are testing a novel vaccine approach. It focuses immune response to the highly conserved sequences surrounding the HIV protease cleavage sites (PCS) to disrupt viral maturation, while limiting excessive immune activation. Our pilot studies using nonhuman primate SIV infection models suggest that this approach is feasible and promising.

Background: Understanding natural HIV control may lead to new preventative or therapeutic strategies. Several protective major histocompatibility complex (MHC) genotypes were found in humans and rhesus macaques. Here, we report a simian immunodeficiency virus (SIV) controller MHC genotype in Mauritian cynomolgus macaques (MCMs).Methods: Twelve MHC-genotyped MCMs were infected with SIVmac251 and monitored for viral loads and CD4(+) T-cell counts.Results: Two macaques with M3M4 genotype exhibited the lowest peak viral loads (log plasma SIV RNA copies/mL), nearly 3 logs lower than those in most macaques with other MHC haplotype combinations, and set point viral loads below the level of detection limit by RT-qPCR (< 2 log RNA copies/mL). They maintained healthy CD4(+) T-cell counts of > 500 cells/mu L blood, while CD4 counts in the vast majority of other macaques were below this level.Conclusions: The M3M4 MHC genotype may confer enhanced control of SIV replication in MCMs.

Background: Human Immunodeficiency Virus (HIV) viral load and CD4 + cell counts are the most commonly used markers for monitoring efficacy of anti-retroviral therapy (ART) in HIV infected individuals. The high cost of viral load monitoring limits its usage in resource limited countries, often leaving the use of CD4+ T cell counts as the only alternative. Though cheaper and more readily available, CD4+ cell counts as a measure of detecting treatment failure, is an unreliable predictor of disease progression. Hence, there is a need for more sensitive alternative, but less costly techniques for detecting treatment failure which can be used in resource limited settings. Objective: To evaluate the feasibility of using plasma CD26/Dipeptidyl peptidase IV (DPPIV) as a novel marker for clinical evaluation of treatment efficacy in HIV infected children. Method: Blood samples collected from HIV + children (n=76) before and after initiation on ART, were assessed for HIV RNA (viral load), CD4+ T-cell count and DPPIV/CD26 levels. Viral load levels were analyzed using Roche Amplicor HIV-1 Monitor Test kit; CD4+ T-Cell Counts were analyzed using BD FACS Calibur flow cytometer while DPPIV/CD 26 levels were analyzed using Human DPPIV/CD26 Quantikine ELISA kit (Ru0026D Systems, Minneapolis MN). Results: The plasma DPPIV/CD26 levels increased significantly in children after ART initiation ( p = 0.017), while the viral load levels declined after ART initiation with subsequent CD4+ cell counts increase. The DPPIV/CD 26 increase positively correlated with viral load decrease while negatively correlating to the CD4+ cell count increase. Conclusion: These findings demonstrate an inverse relationship between DPPIV/CD26 levels and HIV viral load and the direct proportionality of CD4+ Cell counts and DPPIV/CD26 levels, suggesting potential for use of DPPIV/CD26 as a surrogate marker for evaluating HIV disease progression in children receiving anti-retroviral therapy. Key words: CD26/Dipeptidyl peptidase IV (DPPIV), ELISA, Surrogate marker, Viral Load, CD4 Count, antiretroviral.

Nat. Med.; 10.1038/nm.4206; corrected online 19 October 2016 In the version of this article initially published online, Robert Were Omange's name was misspelled in the author list. The original version listed Robert Omage. The error has been corrected in the print, PDF and HTML versions of this article.

Background: Antiretroviral therapy (ART) in resource-limited settings is effective when backed up with adequate clinical, immunological, and virologic monitoring. Undetected, virologic failure results in increased HIV-1 drug resistance mutations (DRMs), morbidity and mortality, or the need for costly second-line and third-line ART.Objective: To evaluate the prevalence, patterns, and risk factors of virologic failure and slow response to ART, among children and adolescents in resource-limited settings in Nairobi, Kenya.Design: A Retrospective study.Setting: The 8 Lea Toto Programme (LTP) Clinics in Dagoretti, Dandora, Kangemi, Kariobangi, Kawangware, Kibera, Mukuru, and Zimmerman areas of Nairobi. Subjects: One hundred and forty-six HIV-infected children and adolescents aged 1 month to 19 years of the LTP in Nairobi Kenya. Medical and demographic data including, HIV-1 viral loads, information on adherence to ART, HIV-1 DRMs and other key determinants of virologic failure, collected over a period of 2 years, was used for this study.Results: A threshold of 1,000 HIV RNA copies/ml was used to determine treatment outcome. The virologic failure rates in this cohort were 43.8% after 6 months, 32.2% after 12 months, 28.8% after 18 months, and 24.0% after 24 months of first-line ART. Twelve (8.2%) of 146 children showed a slow response to ART: they initially failed ART at 12 months, but had treatment success after 18 to 24 months. The rates of virologic rebound were 4 (2.7%) after 18 months and 3 (2.1%) after 24 months of ART. Multivariate Cox proportional hazards regression revealed that children with suboptimal adherence to ART were 37 times more likely to experience virologic failure (P = 0.000003).Conclusions: This study showed that ART implementation in resource-limited settings is effective when regular virologic monitoring, adherence counselling, and HIV-DR testing are available. Secondly, adherence to ART is a strong predictor of treatment outcome for children and adolescents in resourcelimited settings. Therefore, methods of optimizing adherence levels should be explored and implemented.

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research.

A1 Introduction to the 2nd synchronicity forum of GHRI/CHVI-funded Canadian and African HIV prevention and vaccine teams