Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles.

Definition of the specific germline immunoglobulin (Ig) alleles present in an individual is a critical first step to delineate the ontogeny and evolution of antigen-specific antibody responses. Rhesus and cynomolgus macaques are important animal models for pre-clinical studies, with four main sub-groups being used: Indian- and Chinese-origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. We applied the (Ig) gene inference tool IgDiscover and performed extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. There was allelic overlap between Chinese- and Indian-origin rhesus macaques and also between the two macaque species, which is consistent with substantial admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet maintained high individual allelic diversity. These comprehensive databases of germline IGH alleles for rhesus and cynomolgus macaques provide a resource toward the study of B cell responses in these important pre-clinical models.

Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates.

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.

Impact of variants of concern on SARS-CoV-2 viral dynamics in non-human primates.

The impact of variants of concern (VoC) on SARS-CoV-2 viral dynamics remains poorly understood and essentially relies on observational studies subject to various sorts of biases. In contrast, experimental models of infection constitute a powerful model to perform controlled comparisons of the viral dynamics observed with VoC and better quantify how VoC escape from the immune response. Here we used molecular and infectious viral load of 78 cynomolgus macaques to characterize in detail the effects of VoC on viral dynamics. We first developed a mathematical model that recapitulate the observed dynamics, and we found that the best model describing the data assumed a rapid antigen-dependent stimulation of the immune response leading to a rapid reduction of viral infectivity. When compared with the historical variant, all VoC except beta were associated with an escape from this immune response, and this effect was particularly sensitive for delta and omicron variant (p<10-6 for both). Interestingly, delta variant was associated with a 1.8-fold increased viral production rate (p = 0.046), while conversely omicron variant was associated with a 14-fold reduction in viral production rate (p<10-6). During a natural infection, our models predict that delta variant is associated with a higher peak viral RNA than omicron variant (7.6 log10 copies/mL 95% CI 6.8-8 for delta; 5.6 log10 copies/mL 95% CI 4.8-6.3 for omicron) while having similar peak infectious titers (3.7 log10 PFU/mL 95% CI 2.4-4.6 for delta; 2.8 log10 PFU/mL 95% CI 1.9-3.8 for omicron). These results provide a detailed picture of the effects of VoC on total and infectious viral load and may help understand some differences observed in the patterns of viral transmission of these viruses.

Durable immunogenicity, adaptation to emerging variants, and low-dose efficacy of an AAV-based COVID-19 vaccine platform in macaques.

The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo: Potentials and Pitfalls.

For a long while, 8-aminoquinoline compounds have been the only therapeutic agents against latent hepatic malaria parasites. These have poor activity against the blood-stage plasmodia causing acute malaria and must be used in conjunction with partner blood schizontocidal agents. We examined the impacts of one such agent, chloroquine, upon the activity of primaquine, an 8-aminoquinoline, against hepatic stages of Plasmodium cynomolgi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium falciparum within several ex vivo systems-primary hepatocytes of Macaca fascicularis, primary human hepatocytes, and stably transformed human hepatocarcinoma cell line HepG2. Primaquine exposures to formed hepatic schizonts and hypnozoites of P. cynomolgi in primary simian hepatocytes exhibited similar 50% inhibitory concentration (IC50) values near 0.4 µM, whereas chloroquine in the same system exhibited no inhibitory activities. Combining chloroquine and primaquine in this system decreased the observed primaquine IC50 for all parasite forms in a chloroquine dose-dependent manner by an average of 18-fold. Chloroquine also decreased the primaquine IC50 against hepatic P. falciparum in primary human hepatocytes, P. berghei in simian primary hepatocytes, and P. yoelii in primary human hepatocytes. Chloroquine had no impact on primaquine IC50 against P. yoelii in HepG2 cells and, likewise, had no impact on the IC50 of atovaquone (hepatic schizontocide) against P. falciparum in human hepatocytes. We describe important sources of variability in the potentiation of primaquine activity by chloroquine in these systems. Chloroquine potentiated primaquine activity against hepatic forms of several plasmodia. We conclude that chloroquine specifically potentiated 8-aminoquinoline activities against active and dormant hepatic-stage plasmodia in normal primary hepatocytes but not in a hepatocarcinoma cell line.

In Vivo Modulation of Cervicovaginal Drug Transporters and Tissue Distribution by Film-Released Tenofovir and Darunavir for Topical Prevention of HIV-1.

Clinical trials have demonstrated partial protection against HIV-1 infection by vaginal microbicide formulations based on antiretroviral (ARV) drugs. Improved formulations that will maintain sustained drug concentrations at viral target sites in the cervicovaginal mucosa are needed. We have previously demonstrated that treatment of cervicovaginal cell lines with ARV drugs can alter gene expression of drug transporters, suggesting that the mucosal disposition of ARV drugs delivered vaginally can be modulated by drug transporters. This study aimed to investigate in vivo modulation of drug transporter expression in a nonhuman primate model by tenofovir and darunavir released from film formulations. Cervicovaginal tissues were collected from drug-naïve macaques and from macaques vaginally treated with film formulations of tenofovir or darunavir. Drug release in vaginal fluid as well as drug absorption in cervicovaginal tissues and lymph nodes were verified by mass spectrometry. The effects of exposure to drugs on the expression of transporters relevant to ARV drugs were evaluated by quantitative PCR. We showed expression in cervicovaginal tissue of drug-naïve macaques of transporters important for distribution of ARV drugs, albeit at lower levels compared to human tissue for key transporters including P-glycoprotein. Concentrations of tenofovir and darunavir well above the EC50 values determined in vitro were detected in vaginal fluid and vaginal tissues of macaques treated with drug-dissolving films over 24 h and were also comparable to those shown previously to modulate drug transporter expression. Accordingly, Multidrug Resistance associated Protein 2 (MRP2) in cervicovaginal tissue was upregulated by both tenofovir and darunavir. The two drugs also differentially induced and/or inhibited expression of key uptake transporters for reverse transcriptase inhibitors and protease inhibitors. The lower expression of key transporters in macaques may result in increased retention of ARV drugs at the simian cervicovaginal mucosa compared to the human mucosa and has implications for translation of preclinical data. Modulation of drug transporter expression by tenofovir and darunavir points to the potential benefit of MRP2 inhibition to increase ARV drug penetration through the cervicovaginal epithelium.

Vaccination with the Conserved Caveolin-1 Binding Motif in Human Immunodeficiency Virus Type 1 Glycoprotein gp41 Delays the Onset of Viral Infection and Provides Partial Protection in Simian/Human Immunodeficiency Virus-Challenged Cynomolgus Macaques.

We have previously reported that the CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, elicits peptide-specific antibodies. Here, we have investigated the cellular immune response and the protective efficacy against a simian/human immunodeficiency virus (SHIV162P3) challenge. In addition to the CBD1 peptide, peptides overlapping the caveolin-binding-motif (CBM) (622IWNNMTWMQW631 or 622IWNNMTW628) were fused to a Gag-p24 T helper epitope for vaccination. All immunized cynomolgus macaques responded to a cocktail peptide immunization by inducing specific T cells and the production of high-titer CBD1/CBM peptide-specific antibodies. Six months after the fourth vaccine boost, six control and five vaccinated animals were challenged weekly by repeated exposure to SHIV162P3 via the mucosal rectal route. All control animals were infected after 1 to 3 challenges with SHIV, while among the five vaccinated monkeys, three became infected after a delay compared to control; one was infected after the eighth viral challenge, and one remained uninfected even after the ninth SHIV challenge. Immunized animals maintained a CD4 T cell count, and their central memory CD4 T cells were less depleted than in the control group. Furthermore, SHIV challenge stimulates antigen-specific memory T cell response in vaccinated macaques. Our results indicate that peptides derived from the CBM region can be immunogenic and provide protection against SHIV infection in cynomolgus monkeys.IMPORTANCE In HIV-1-producing cells, gp41 exists in a complexed form with caveolin-1, an interaction most probably mediated by the caveolin-1 binding motif. This sequence is highly conserved in every single HIV-1 isolate, thus suggesting that there is constant selective pressure to preserve this sequence for a specific function in the HIV infectious cycle. Consequently, the CBM sequence may represent the "Achilles' heel" of HIV-1 in the development of an efficient vaccine. Our results demonstrate that macaques immunized with the CBM-based peptides displayed a delay in the onset of viral infection and CD4 depletion, as well as a significant induction of antigen-specific memory T cell response, which is essential for the control of HIV/SIV infections. Finally, as HIV-infected individuals lack anti-CBM immune responses, CBM-based vaccines could have applications as a therapeutic vaccine in AIDS patients.

Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs.

The sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the human immunodeficiency virus (HIV)/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV-infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. In this study, we used single-genome amplification, amplicon sequencing (env gene), and phylogenetic analyses to compare the genetic structures of simian immunodeficiency virus (SIV) populations across all the male genital organs and blood in intravenously inoculated cynomolgus macaques in the chronic stage of infection. Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles, and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens, and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or proinflammatory cytokine mRNA levels. These data indicate that rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.IMPORTANCE Semen is instrumental for the dissemination of viruses through sexual contacts. Worryingly, a number of systemic viruses, such as HIV, can persist in this body fluid in the absence of viremia. The local source(s) of virus in semen, however, remains unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single-genome amplification, amplicon sequencing (env gene), and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them (i.e., from epididymis, vas deferens, and seminal vesicles) displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract.

Modified Vaccinia Virus Ankara Vector Induces Specific Cellular and Humoral Responses in the Female Reproductive Tract, the Main HIV Portal of Entry.

The female reproductive tract (FRT) is one of the major mucosal invasion sites for HIV-1. This site has been neglected in previous HIV-1 vaccine studies. Immune responses in the FRT after systemic vaccination remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized specific immune responses in all compartments of the FRT of nonhuman primates after systemic vaccination. Memory T cells were preferentially found in the lower tract (vagina and cervix), whereas APCs and innate lymphoid cells were mainly located in the upper tract (uterus and fallopian tubes). This compartmentalization of immune cells in the FRT was supported by transcriptomic analyses and a correlation network. Polyfunctional MVA-specific CD8+ T cells were detected in the blood, lymph nodes, vagina, cervix, uterus, and fallopian tubes. Anti-MVA IgG and IgA were detected in cervicovaginal fluid after a second vaccine dose. Thus, systemic vaccination with an MVA vector elicits cellular and Ab responses in the FRT.

Early SIV and HIV infection promotes the LILRB2/MHC-I inhibitory axis in cDCs.

Classical dendritic cells (cDCs) play a pivotal role in the early events that tip the immune response toward persistence or viral control. In vitro studies indicate that HIV infection induces the dysregulation of cDCs through binding of the LILRB2 inhibitory receptor to its MHC-I ligands and the strength of this interaction was proposed to drive disease progression. However, the dynamics of the LILRB2/MHC-I inhibitory axis in cDCs during early immune responses against HIV are yet unknown. Here, we show that early HIV-1 infection induces a strong and simultaneous increase of LILRB2 and MHC-I expression on the surface of blood cDCs. We further characterized the early dynamics of LILRB2 and MHC-I expression by showing that SIVmac251 infection of macaques promotes coordinated up-regulation of LILRB2 and MHC-I on cDCs and monocytes/macrophages, from blood and lymph nodes. Orientation towards the LILRB2/MHC-I inhibitory axis starts from the first days of infection and is transiently induced in the entire cDC population in acute phase. Analysis of the factors involved indicates that HIV-1 replication, TLR7/8 triggering, and treatment by IL-10 or type I IFNs increase LILRB2 expression. Finally, enhancement of the LILRB2/MHC-I inhibitory axis is specific to HIV-1 and SIVmac251 infections, as expression of LILRB2 on cDCs decreased in naturally controlled chikungunya virus infection of macaques. Altogether, our data reveal a unique up-regulation of LILRB2 and its MHC-I ligands on cDCs in the early phase of SIV/HIV infection, which may account for immune dysregulation at a critical stage of the anti-viral response.

Different Expression of Interferon-Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells Based on Their Maturation State.

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence.

Laser capture microdissection enables transcriptomic analysis of dividing and quiescent liver stages of Plasmodium relapsing species.

Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.

Differential activity of methylene blue against erythrocytic and hepatic stages of Plasmodium.

BACKGROUND: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages. METHODS: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice. RESULTS: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes. CONCLUSION: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites.

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis.

Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20(+) B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27(hi)CD21(lo) activated memory and CD27(+)CD21(+) resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.

Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848.

Superior Efficacy of a Human Immunodeficiency Virus Vaccine Combined with Antiretroviral Prevention in Simian-Human Immunodeficiency Virus-Challenged Nonhuman Primates.

UNLABELLED: Although vaccines and antiretroviral (ARV) prevention have demonstrated partial success against human immunodeficiency virus (HIV) infection in clinical trials, their combined introduction could provide more potent protection. Furthermore, combination approaches could ameliorate the potential increased risk of infection following vaccination in the absence of protective immunity. We used a nonhuman primate model to determine potential interactions of combining a partially effective ARV microbicide with an envelope-based vaccine. The vaccine alone provided no protection from infection following 12 consecutive low-dose intravaginal challenges with simian-HIV strain SF162P3, with more animals infected compared to naive controls. The microbicide alone provided a 68% reduction in the risk of infection relative to that of the vaccine group and a 45% reduction relative to that of naive controls. The vaccine-microbicide combination provided an 88% reduction in the per-exposure risk of infection relative to the vaccine alone and a 79% reduction relative to that of the controls. Protected animals in the vaccine-microbicide group were challenged a further 12 times in the absence of microbicide and demonstrated a 98% reduction in the risk of infection. A total risk reduction of 91% was observed in this group over 24 exposures (P = 0.004). These important findings suggest that combined implementation of new biomedical prevention strategies may provide significant gains in HIV prevention. IMPORTANCE: There is a pressing need to maximize the impact of new biomedical prevention tools in the face of the 2 million HIV infections that occur each year. Combined implementation of complementary biomedical approaches could create additive or synergistic effects that drive improved reduction of HIV incidence. Therefore, we assessed a combination of an untested vaccine with an ARV-based microbicide in a nonhuman primate vaginal challenge model. The vaccine alone provided no protection (and may have increased susceptibility to a simian-HIV vaginal challenge), while the microbicide reduced the infection risk compared to that of vaccinated and naive animals. Importantly, the combined interventions provided the greatest level of protection, which was sustained following withdrawal of the microbicide. The data suggest that provision of ARV prophylaxis during vaccination reduces the potential for unexpected increased risks of infection following immunization and augments vaccine efficacy. These findings are important for the potential adoption of ARV prophylaxis as the baseline intervention for future HIV/AIDS vaccines.