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The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a β-hairpin linear epitope. In vitro , NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIV mac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIV mac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14 + efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44 + cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIV mac251 acquisition, highlighting the complexity of antibody responses to V2.

Weather-related disasters are increasing in frequency and severity, leaving survivors to cope with ensuing mental, financial, and physical hardships. This adversity can exacerbate existing morbidities, trigger new ones, and increase the risk of mortality-features that are also characteristic of advanced age-inviting the hypothesis that extreme weather events may accelerate aging. To test this idea, we examined the impact of Hurricane Maria and its aftermath on immune cell gene expression in large, age-matched, cross-sectional samples from free-ranging rhesus macaques ( Macaca mulatta ) living on an isolated island. A cross section of macaques was sampled 1 to 4 y before ( n = 435) and 1 y after ( n = 108) the hurricane. Hurricane Maria was significantly associated with differential expression of 4% of immune-cell-expressed genes, and these effects were correlated with age-associated alterations in gene expression. We further found that individuals exposed to the hurricane had a gene expression profile that was, on average, 1.96 y older than individuals that were not-roughly equivalent to an increase in 7 to 8 y of a human life. Living through an intense hurricane and its aftermath was associated with expression of key immune genes, dysregulated proteostasis networks, and greater expression of inflammatory immune cell-specific marker genes. Together, our findings illuminate potential mechanisms through which the adversity unleashed by extreme weather and potentially other natural disasters might become biologically embedded, accelerate age-related molecular immune phenotypes, and ultimately contribute to earlier onset of disease and death., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing., Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques., Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test., Interpretation: Taken together, the COVID-19 NAb-test TM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care., Funding: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation., Competing Interests: Declaration of Competing Interest A provisional patent covering the COVID-19 NAb-test(TM) test and underlying technology has been submitted through The University of Melbourne. Dr. Fulford reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Van reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Mr. Van has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Dr. Gherardin reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Ms. Zheng reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Ms. Zheng has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Ciula has nothing to disclose. Prof. Drummer has nothing to disclose. Mr. Redmond has nothing to disclose. Dr. Tan has nothing to disclose. Ms. Boo has nothing to disclose. Dr. Center has nothing to disclose. Dr. Li has nothing to disclose. Dr. Grimley has nothing to disclose. Dr. Wines reports grants from Australian Govt, Medical Research Future Fund, grants from NHMRC, National Health and Medical Research Council GNT1145303, Australia, during the conduct of the study; In addition, Dr. Wines has a patent drafted for use of ACE2-Fc pending. Dr. Nguyen has nothing to disclose. Ms. Mordant has nothing to disclose. Dr. Ellenberg has nothing to disclose. Dr. Rowntree has nothing to disclose. Lukasz Kedzierski has nothing to disclose. Prof. Cheng reports that he is a member of government advisory committees advising on COVID policy. Prof. Doolan has nothing to disclose. Prof. Matthews reports grants from Curran Foundation, during the conduct of the study; grants from Gilead sciences, grants from Abbvie Inc outside the submitted work. Dr. Bond has nothing to disclose. Prof. Hogarth reports grants from NHMRC, grants from MRFF, during the conduct of the study; In addition, Prof. Hogarth has a patent Antiviral pending. A/Prof. McQuilten reports grants from Medical Research Future Fund, during the conduct of the study. Prof. Subbarao has nothing to disclose. Prof. Kedzierska has nothing to disclose. Dr. Juno has nothing to disclose. Dr. Wheatley has nothing to disclose. Prof. Kent has nothing to disclose. Prof. Williamson has nothing to disclose. Prof. Purcell reports grants from Victorian Government DHHS, grants from Medical Research Future Fund, other from Jack Ma Foundation, during the conduct of the study; In addition, Prof. Purcell has a patent PCT/AU2021/050839 pending. Prof. Anderson reports grants from Government of the State of Victoria, during the conduct of the study; other from Nanjing BioPoint Diagnostic Technology, outside the submitted work; In addition, Prof. Anderson has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Prof. Godfrey reports grants from Victorian Government DHHS, and from Australian Research Council, during the conduct of the study; grants from Medical Research Future Fund, other from Jack Ma Foundation, grants from National Health and Medical Research Council, outside the submitted work; In addition, Prof. Godfrey has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending., (Copyright © 2021. Published by Elsevier B.V.)

Background: The recent ongoing outbreak of coronavirus disease 2019 (COVID-19), still is an unsolved problem with a growing rate of infected cases and mortality worldwide. The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is targeting the angiotensin-converting enzyme 2 (ACE2) receptor and mostly causes a respiratory illness. Although acquired and resistance immunity is one of the most important aspects of alleviating the trend of the current pandemic; however, there is still a big gap of knowledge regarding the infection process, immunopathogenesis, recovery, and reinfection., Aim of Review: To answer the questions regarding "the potential and probability of reinfection in COVID-19 infected cases" or "the efficiency and duration of SARS-CoV-2 infection-induced immunity against reinfection" we critically evaluated the current reports on SARS-CoV-2 immunity and reinfection with special emphasis on comparative studies using animal models that generalize their finding about protection and reinfection. Also, the contribution of humoral immunity in the process of COVID-19 recovery and the role of ACE2 in virus infectivity and pathogenesis has been discussed. Furthermore, innate and cellular immunity and inflammatory responses in the disease and recovery conditions are reviewed and an overall outline of immunologic aspects of COVID-19 progression and recovery in three different stages are presented. Finally, we categorized the infected cases into four different groups based on the acquired immunity and the potential for reinfection., Key Scientific Concepts of Review: In this review paper, we proposed a new strategy to predict the potential of reinfection in each identified category. This classification may help to distribute resources more meticulously to determine: who needs to be serologically tested for SARS-CoV-2 neutralizing antibodies, what percentage of the population is immune to the virus, and who needs to be vaccinated., Competing Interests: The authors declare that they have no actual or potential conflict of interests., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2) 1-4 . Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID 50 ) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3α genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized 1 and 6 month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.

The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in <20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19., Competing Interests: Declaration of Competing Interest The authors declare that they are affiliated with up to two of four commercial entities with stakes in the data., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The major histocompatibility complex (MHC) class I-related molecule, MR1, presents vitamin B metabolites from bacteria and yeast to mucosal-associated invariant T (MAIT) cells. Despite the evolutionary conservation of MR1, we do not know whether different allele variants of MR1 exist within the nonhuman primate (NHP) populations that are commonly used for biomedical research. In this study, we identified 21 distinct MR1 nucleotide sequences representing 32 different alleles across five different NHP populations. The majority of the alleles conferring amino acid changes (allele variants) were found in or near the alpha-1 domain of the mature MR1 protein. We expressed four of the most commonly observed MR1 allele variants in 293T cells, and we found that each variant could present bacterial metabolites on the cell surface. We successfully induced cytokine production in macaque MAIT cells stimulated with 293T cells expressing the four most common MR1 allele variants, demonstrating the usefulness of these cell lines to study MAIT cell activity. Our data suggests that MR1 is not monomorphic, but that there are multiple MR1 alleles in NHPs. The materials we describe here will be valuable for characterizing differences in MR1 antigen presentation and MAIT cell function in NHPs.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli., Competing Interests: The authors have declared that no competing interests exist.

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A (TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera (amTRIMCyp), along with a TRIM5α allelic protein (amTRIM5α). Herein, we investigated the antiviral activity of amTRIMCyp and amTRIM5α individually, as well as their interaction and joint effects. amTRIMCyp showed a divergent restriction pattern from amTRIM5α. Although both proteins potently restricted the replication of HIV-1, only amTRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when amTRIMCyp and amTRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous amTRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from amTRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the amTRIMCyp-amTRIM5α interaction. In contrast, amTRIMCyp completely abrogated the anti-N-MLV activity mediated by amTRIM5α, showing a dominant-negative effect, indicating that the generation of amTRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.

Structural similarities between Zika (ZIKV) and dengue virus (DENV) leads to the induction of cross-reactive responses. We have previously demonstrated that ZIKV exposed macaques significantly enhance DENV viremia. Here we show that this enhancement of DENV infection occurred in the presence of high levels of DENV cross-reactive IgG1 subclass of binding antibodies (bAb) with low DENV neutralizing antibody (nAb) activity (<1:10). The DENV-2 nAb titres after ZIKV infection were, however, higher than those induced in DENV-2 only infected animals suggesting that ZIKV induced low titres of cross-nAb against DENV. Surprisingly, DENV-2 infection of animals previously infected with ZIKV was not accompanied by an anamnestic increase in cross-nAb titres till about 1 week after DENV-2 infection. This delay coincided with enhanced DENV-2 viremia indicating that high levels of cross-bAb in the absence of high nAb contributes to enhancement of DENV infection. Serum collected 8 weeks after DENV-2 infection had high levels of nAb and showed delayed antibody dependent enhancement (ADE) of infection (1:100 dilution) as compared with serum that was collected from ZIKV infected animals prior to DENV-2 infection (1:10 dilution). Examination of serum from macaques that were simultaneously infected with both ZIKV and DENV-2 showed high levels of nAb and delayed ADE responses raising the possibility that the low levels of cross-nAb induced by ZIKV infection could be overcome by co-immunization against ZIKV and DENV infection. Taken together, our results provide additional insights into the nature and kinetics of cross-reactive antibody responses and identify a critical correlate that could potentially prevent enhancement of DENV infection during ZIKV convalescence.

The Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1β (IL-1β) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1β and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans., (Copyright © 2018 Patton et al.)

Parasites can increase infection rates and pathogenicity in immunocompromised human immunodeficiency virus (HIV) patients. However, in vitro studies and epidemiological investigations also suggest that parasites might escape immunocompromised hosts during HIV infection. Due to the lack of direct evidence from animal experiments, the effects of parasitic infections on immunocompromised hosts remain unclear. Here, we detected 14 different parasites in six northern pig-tailed macaques (NPMs) before or at the 50th week of simian immunodeficiency virus (SIV) infection by ELISA. The NPMs all carried parasites before viral injection. At the 50th week after viral injection, the individuals with negative results in parasitic detection (i.e., 08247 and 08287) were characterized as the Parasites Exit (PE) group, with the other individuals (i.e., 09203, 09211, 10205, and 10225) characterized as the Parasites Remain (PR) group. Compared with the PR group, the NPMs in the PE group showed higher viral loads, lower CD4 + T cells counts, and lower CD4/CD8 rates. Additionally, the PE group had higher immune activation and immune exhaustion of both CD4 + and CD8 + T cells. Pathological observation showed greater injury to the liver, cecum, colon, spleen, and mesenteric lymph nodes in the PE group. This study showed more seriously compromised immunity in the PE group, strongly indicating that parasites might exit an immunocompromised host.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi - macaque model systems and summarizing exciting new progress in this area of research.

Background: The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to Streptococcus pneumoniae., Methods: We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC) or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS) using microengraving (a single-cell analysis method) and single-cell RT-PCR., Results: Seven days after boosting, the mean frequency of antigen-specific memory B cells was significantly increased in macaques vaccinated with 13vPnC compared to those receiving 23vPS. The 13vPnC-vaccinated macaques also exhibited a more even distribution of antibody specificities to four polysaccharides in the vaccine (PS4, 6B, 14, 23F) that were examined. However, single-cell analysis of the antibody variable region sequences from antigen-specific B cells elicited by unconjugated and conjugated vaccines indicated that both the germline gene segments forming the heavy chains and the average lengths of the Complementary Determining Region 3 (CDR3) were similar., Conclusions: Our results confirm that distinctive differences can manifest between antigen-specific memory B cell repertoires in nonhuman primates immunized with conjugated and unconjugated pneumococcal polysaccharide vaccines. The study also supports the notion that the conjugated vaccines have a favorable profile in terms of both the frequency and breadth of the anamnestic response among antigen-specific memory B cells.

Ten novel MHC-DPB1 alleles of Tibetan macaque, were identified by cloning and sequencing., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4β7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity., Competing Interests: AS is the CEO of Oncovir, which is developing polyICLC (Hiltonol), and is both employed by and owns stock in the company. The authors have no other competing interests. We confirm that our statement concerning AS, the CEO of Oncovir, does not alter our adherence to PLOS ONE policies on sharing data and materials.

Virus-specific CD8(+) T cells exert strong suppressive pressure on human/simian immunodeficiency virus (HIV/SIV) replication. These responses have been intensively examined in peripheral blood mononuclear cells (PBMCs) but not fully analyzed in lymph nodes (LNs), where interaction between CD8(+) T cells and HIV/SIV-infected cells occurs. Here, we investigated target antigen specificity of CD8(+) T cells in LNs in a macaque AIDS model. Analysis of virus antigen-specific CD8(+) T-cell responses in the inguinal LNs obtained from twenty rhesus macaques in the chronic phase of SIV infection showed an inverse correlation between viral loads and frequencies of CD8(+) T cells with CD28(+) CD95(+) central memory phenotype targeting the N-terminal half of SIV core antigen (Gag-N). In contrast, analysis of LNs but not PBMCs revealed a positive correlation between viral loads and frequencies of CD8(+) T cells with CD28(-)CD95(+) effector memory phenotype targeting the N-terminal half of SIV envelope (Env-N), soluble antigen. Indeed, LNs with detectable SIV capsid p27 antigen in the germinal center exhibited significantly lower Gag-N-specific CD28(+) CD95(+) CD8(+) T-cell and higher Env-N-specific CD28(-)CD95(+) CD8(+) T-cell responses than those without detectable p27. These results imply that core and envelope antigen-specific CD8(+) T cells show different patterns of interactions with HIV/SIV-infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory diseases. Because anti-HTLV-1 immune responses are critical for suppressing infected cells, enhancing cellular immunity is beneficial for the treatment of HTLV-1-associated diseases. Using simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaques, we analyzed the immune responses to viral antigens and the dynamics of virus-infected cells. The chemokine receptor CCR4 is expressed on STLV-1 infected cells, and administration of humanized monoclonal antibody to CCR4, mogamulizumab, dramatically decreased the number of STLV-1-infected cells in vivo. Concurrently, mogamulizumab treatment enhanced STLV-1 specific CD4(+) and CD8(+) T cell responses by simultaneously targeting CCR4(+) effector regulatory T (Treg) cells and infected cells. Mogamulizumab promoted the phagocytosis of CCR4(+) infected cells by macrophages, which likely enhanced antigen presentation. Vaccination with recombinant vaccinia virus (rVV) expressing viral antigens suppressed the proviral load and the number of Tax-expressing cells. Enhanced T-cell responses were also observed in some ATL patients who were treated with mogamulizumab. This study shows that mogamulizumab works not only by killing CCR4(+) infected cells directly, but also by enhancing T cell responses by increasing the phagocytosis of infected cells by antigen-presenting cells and suppressing CCR4(+) effector Treg cells.

Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368-606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques' sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses.

The northern pig-tailed macaque (Macaca leonina) has been confirmed to be an independent species from the pig-tailed macaque group of Old World monkey. We have previously reported that the northern pig-tailed macaques were also susceptible to HIV-1. Here, to make this animal a potential HIV/AIDS model and to discover the mechanism of virus control, we attempted to assess the role of major histocompatibility complex (MHC) class I-restricted immune responses to HIV-1 infection, which was associated with viral replication and disease progression. As an initial step, we first cloned and characterized the classical MHC class I gene of northern pig-tailed macaques. In this study, we identified 39 MHC class I alleles including 17 MHC-A and 22 MHC-B alleles. Out of these identified alleles, 30 were novel and 9 were identical to alleles previously reported from other macaque species. The MHC-A and MHC-B loci were both duplicates as rhesus macaques and southern pig-tailed macaques. In addition, we also detected the patterns of positive selection in northern pig-tailed macaques and revealed the existence of balance selection with 20 positive selection sites in the peptide binding region. The analysis of B and F peptide binding pockets in northern and southern pig-tailed macaques and rhesus macaques suggested that they were likely to share a few common peptides to present. Thus, this study provides important MHC immunogenetics information and adds values to northern pig-tailed macaques as a promising HIV/AIDS model.

With the unprecedented scale of the 2014-2016 West Africa outbreak, the clinical and scientific community scrambled to identify potential therapeutics for Ebola virus disease (EVD). Passive administration of antibodies has a long successful history for prophylaxis and therapy of a variety of infectious diseases, but the importance of antibodies in EVD has been unclear and is the subject of some debate. Recent studies in non-human primates have renewed interest in the potential of antibodies to impact EVD. Currently ongoing clinical evaluation of polyclonal and monoclonal antibody therapy in EVD patients in West Africa may finally offer a definitive answer to this debate., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classic human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provide a basis for defining immediate post-injection vaccine responses in primates., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The 2005 outbreak of Marburg virus (MARV) infection in Angola was the most lethal MARV infection outbreak in history, with a case-fatality rate (90%) similar to that for Zaire ebolavirus (EBOV) infection. However, very little is known about the pathogenicity of MARV Angola, as few studies have been conducted to date. Therefore, the immune response was examined in MARV Angola-infected nonhuman primates., Methods: Cynomolgus macaques were infected with MARV Angola and monitored for survival. The effect of MARV Angola on the immune system was examined by immunophenotyping whole-blood and by analyzing cytokine and chemokine levels in plasma and spleen specimens, using flow cytometry., Results: The prominent clinical findings were rapid onset of disease and death (mean time after infection, 6.7 days), fever, depression, anorexia, petechial rash, and lymphopenia. Specifically, T, B, and natural killer cells were severely depleted in the blood by day 6. The typical cytokine storm was present, with levels of interferon γ, tumor necrosis factor, interleukin 6, and CCL2 rising in the blood early during infection., Conclusions: MARV Angola displayed the same virulence and disease pathology as EBOV. MARV Angola appears to cause a more rapid onset and severe outcome of infection than other MARV strains., (© Crown copyright 2015.)

Natural killer (NK) cells play a central role in immune responses through direct cytotoxicity and the release of cytokines that prime adaptive immunity. In simian primates, NK cell responses are regulated by interactions between two highly polymorphic sets of molecules: the killer-cell immunoglobulin-like receptors (KIRs) and their major histocompatibility complex (MHC) class I ligands. KIR-MHC class I interactions in humans have been implicated in the outcome of a number viral diseases and cancers. However, studies to address the role of KIRs in animal models have been limited by the complex immunogenetics and lack of defined ligands for KIRs in non-human primates. Due to the rapid evolution of KIRs, there is little conservation among the KIR genes of different primate species and it is not possible to predict the specificity of KIRs from known KIR-MHC class I interactions in humans. Hence, the MHC class I ligands for KIRs in species other than humans are poorly defined. Here, we review the KIR genes of the rhesus macaque, an important animal model for human immunodeficiency virus infection and other infectious diseases, and the MHC class I ligands that have been identified for KIRs in this species., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although the human malaria parasite Plasmodium vivax is closely related to Asian Old World monkey malaria parasites, there are no reports of P. vivax infections in macaques. In this study, we compared the infectivity of P. vivax and Plasmodium cynomolgi in Japanese macaques (Macaca fuscata) and in cynomolgus macaques (Macaca fascicularis). The Japanese macaques were highly susceptible to P. cynomolgi but not to P. vivax, whereas cynomolgus macaques showed mild/limited P. cynomolgi infection and were, also, not susceptible to P. vivax. Serotyping and amino acid sequence comparison of erythrocyte surface Duffy antigen/receptor for chemokines (DARC) indicate that the Japanese macaque DARC sequence is nearly identical to that of rhesus (Macaca mulatta) and cynomolgus macaques. This suggests that the macaques share a common mechanism for preventing P. vivax infection. Comparison of amino acid sequences of the Duffy-binding-like (DBL) domain from several different Plasmodium species suggests that P. vivax DBLs will not bind to macaque DARCs, which can explain the lack of P. vivax infectivity. The DBL sequence analyses also suggest that P. cynomolgi DBLs may target Japanese macaque erythrocytes through a DARC-independent interaction., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

Minimal conditioning or even no conditioning would be the preferred preparation for most gene therapy applications for nonmalignant diseases. However, reduced intensity conditioning (RIC) regimens in patients with nonhematologic malignancies have not led to long-term engraftment unless a selective advantage was present for the transplanted donor cells. Similar findings have also been observed in a number of large animal studies. Inadequate myelosuppression levels were thought to be responsible for the outcomes. To address this issue several innovative protocols in small animals have been presented with selective hematopoietic myelosuppression and less systemic toxicity. Such protocols promised to curb the transplant-related morbidity and mortality in myeloablative conditioning and provide effective long-term engraftment, especially in patients with gene-corrected autografts. In the present study we have tested some of these promising RIC regimens in nonhuman primates, a clinically relevant large animal model. Our data suggest that transient myelosuppression induced by anti-c-Kit antibody in conjunction with low-dose irradiation may lead to long-term engraftment, albeit at low levels. The animals with busulfan conditioning with or without anti-c-Kit that received gene-modified autologous transplants with green fluorescent protein expression had similar myelosuppression, but failed long-term engraftment and despite immunosuppressive treatment had all the hallmarks seen previously in similar models without immunosuppression. Our preliminary data expand current knowledge of RIC and emphasize the need to explore whether specific and directed myelosuppression alone is adequate in the absence of microenvironmental modulation, or whether innovative combinations are necessary for safe and effective engraftment.

Background: A small percentage of human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected macaques control virus replication without antiretroviral treatment. The major determinant of this control is host expression of certain major histocompatibility complex alleles. However, this association is incompletely penetrant, suggesting that additional loci modify the major histocompatibility complex's protective effect. Here, to identify candidate control-modifying loci, we sequence the genomes of 12 SIV-infected Mauritian cynomolgus macaques that experienced divergent viral load set points despite sharing the protective M1 major histocompatibility complex haplotype., Results: Our genome-wide analysis of haplotype-level variation identifies seven candidate control-modifying loci on chromosomes 2, 3, 7, 8, 9, 10, and 14. The highest variant density marks the candidate on chromosome 7, which is the only control-modifying locus to comprise genes with known immunological function. Upon closer inspection, we found an allele for one of these genes, granzyme B, to be enriched in M1(+) controllers. Given its established role as a cytotoxic effector molecule that participates in CD8-mediated killing of virus-infected cells, we test the role of variation within gzmb in modifying SIV control by prospectively challenging M1(+) granzyme B-defined macaques., Conclusions: Our study establishes a framework for using whole genome sequencing to identify haplotypes that may contribute to complex clinical phenotypes. Further investigation into the immunogenetics underlying spontaneous HIV control may contribute to the rational design of a vaccine that prevents acquired immune deficiency syndrome.

Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.

Unlabelled: Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome., Importance: Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Little is known about the effects of Major Histocompatibility Complex (MHC) haplotypes on immunity to primate lentiviruses involving both acquired and innate immune responses. We present statistical evidence of the influence of MHC polymorphism on antiviral immunity of Mauritian cynomolgus macaques (MCM) following simian/human immunodeficiency virus SHIVSF162P4cy infection, involving the production of pro- and anti-inflammatory cytokines and α-defensins, which may modulate acquired immune responses. During the acute phase of infection, IL-10 correlated positively with viral load and negatively with CD4+T cell counts. Furthermore, α-defensins production was directly correlated with plasma viral RNA, particularly at peak of viral load. When the effects of the MHC were analyzed, a significant association between lower anti-Env binding and neutralizing antibody levels with class IB M4 haplotype and with class IA, IB M4 haplotype, respectively, was observed in the post-acute phase. Lower antibody responses may have resulted into a poor control of infection thus explaining the previously reported lower CD4 T cell counts in these monkeys. Class II M3 haplotype displayed significantly lower acute and post-acute IL-10 levels. In addition, significantly lower levels of α-defensins were detected in class IA M3 haplotype monkeys than in non-M3 macaques, in the post-acute phase of infection. These data indicate that the MHC could contribute to the delicate balance of pro-inflammatory mechanisms, particularly with regard to the association between IL-10 and α-defensins in lentivirus infection. Our results show that host genetic background, virological and immunological parameters should be considered for the design and interpretation of HIV-1 vaccine efficacy studies.

The major histocompatibility complex (MHC) plays an important role in the immune response and may thus crucially affect an individual's fitness, relevant also for studies on evolutionary ecology and wildlife conservation. Detailed knowledge on the genomic organization, polymorphism and diversity of the MHC has a narrow taxonomic focus though and among macaques is only available for rhesus and long-tailed macaques-the species most commonly kept for biomedical research. The lack of data on wild populations is largely due to the difficulty of obtaining blood or tissue samples necessary for genotyping approaches. Here, we aimed at analyzing MHC-DRB from non-invasively collected fecal samples in wild Assamese macaques (Macaca assamensis), utilizing the MHC-DRB-STR (D6S2878) microsatellite marker. Due to the fecal DNA source incomplete genotypes occurred, which may be improved in the future by method refinement. We detected 28 distinct DRB-STR lengths in 43 individuals with individual genotypes containing 1-9 MHC-DRB-STRs and defined four haplotypes segregating between families in Mendelian fashion. Our results indicate that variability and diversity of MHC-DRB in Assamese macaques is comparable to that of other macaque species and importantly, that fecal samples can be used for non-invasive analysis of MHC genes after refinement of the applied methods, opening a number of opportunities for MHC research on natural populations., (© 2013 Wiley Periodicals, Inc.)

The rodent testis is well established as a site of immune privilege where both innate and acquired immune responses are suppressed. Immune cells and responses within human or non-human primate testes, by contrast, are poorly characterised. This study used multi-colour flow cytometry to characterise the leukocytes in testicular cells isolated from 12 young adult pigtail macaques (Macaca nemestrina) by collagenase dispersal, and to measure the cytokine responses of macaque testicular T-lymphocytes to mitogens. B-lymphocytes and granulocytes were present in very low numbers (0.24% and 3.3% of leukocytes respectively), indicating minimal blood contamination. A median of 30.8% of the recovered testicular leukocytes were CD3+ lymphocytes, with CD4 and CD8 T-lymphocyte proportions similar to those in the blood. The proportion of naïve T-lymphocytes in the testis was low, with significantly higher frequencies of central memory cells, compared with the blood. A median of 42.7% of the testicular leukocytes were CD163+ macrophages, while 4.5% were CD14+CD163- monocyte-like macrophages. Small populations of myeloid and plasmacytoid dendritic cells, NK cells and NKT cells were also detected. Following mitogen stimulation, 19.7% of blood T-lymphocytes produced IFNγ and/or TNF, whereas significantly fewer (4.4%) of the testicular T-lymphocytes responded to stimulation. Our results characterise the immune cells within the adult macaque testis and identify a suppression of T-lymphocyte responses. This study provides a baseline to examine the immunology of the primate testis and suggests that testicular immune privilege could also be present in primates., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Tibetan macaques (Macaca thibetana), stump-tailed macaques (M. arctoides), Assamese macaques (M. assamensis), and northern pig-tailed macaques (M. leonina) are four major species of Macaca in China. In order to effectively use these species in biomedical research, thorough investigations of their MHC immunogenetics are required. In this study, we identified MHC class I sequences using cDNA cloning and sequencing on a cohort of six M. thibetana, three M. arctoides, three M. assamensis, and three M. leonina derived from Sichuan and Yunnan provinces of China. Eighty new alleles were identified, including 26 MHC-A alleles, 46 MHC-B alleles, and 8 MHC-I alleles. Among them, Math-A1*126:01, Math-B*190:01, Math-B*191:01, Math-B*192:01, Maar-A1*127:01, Maar-A1*129:01, and Maas-A1*128:01 represent lineages that had not been reported earlier in Macaca. Phylogenetic analyses show that no obvious separation of lineages among these species of Macaca. This study provides important information about the MHC immunogenetics for the four major species of Chinese macaques and adds value to these species as model organisms in biomedical research.

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9-14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5 weeks in some long-term chronically SHIV-infected animals with low CD4(+) T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

ZMAb is a promising treatment against Ebola virus (EBOV) disease that has been shown to protect 50% (two of four) of nonhuman primates (NHPs) when administered 2 days post-infection (dpi). To extend the treatment window and improve protection, we combined ZMAb with adenovirus-vectored interferon-α (Ad-IFN) and evaluated efficacy in EBOV-infected NHPs. Seventy-five percent (three of four) and 100% (four of four) of cynomolgus and rhesus macaques survived, respectively, when treatment was initiated after detection of viremia at 3 dpi. Fifty percent (two of four) of the cynomolgus macaques survived when Ad-IFN was given at 1 dpi, followed by ZMAb starting at 4 dpi, after positive diagnosis. The treatment was able to suppress viremia reaching ~10(5) TCID50 (median tissue culture infectious dose) per milliliter, leading to survival and robust specific immune responses. This study describes conditions capable of saving 100% of EBOV-infected NHPs when initiated after the presence of detectable viremia along with symptoms.

The induction of a robust neutralizing antibody (nAb) response is likely to be as essential as specific cell-mediated immunity (CMI) against multiple antigens for the development of effective preventive and therapeutic vaccines against hepatitis C virus (HCV) infection in humans. To date, no data on the immunogenicity of the replication-defective vaccinia virus (derived from the Tiantan strain) (rNTV)-based HCV vaccine in primates have been reported. This study describes in detail the immunogenicity of various vaccine candidates in rhesus macaques, including rNTV-based and replication-defective recombinant adenoviral (rAd)-based HCV vaccines, as well as HCV pseudotyped virus-like particles (HCVpp). Our data showed that rAd-HCV vaccine boosting induced robust CMI, while priming or boosting with HCVpp enhanced the antigen-specific nAb response after rAd-HCV vaccination; however, CMI was not enhanced. Vaccination includes rNTV-HCV priming induced robust antigen-specific antibody, particularly nAbs, and CMI responses. Furthermore, more robust and longer-lasting CMI and higher cytokine levels (both Th1 and Th2 types, especially IFN-γ) resulted from boosting with rAd-HCV. We conclude that the rNTV-based HCV vaccine induces robust nAbs and CMI when combined with a heterogeneous primer-booster strategy, which shows promise for development of a human HCV vaccine.

Tibetan macaque (Macaca thibetana), an endangered primate species endemic to China, have been used as experimental animal model for various human diseases. Major histocompatibility complex (MHC) genes play a crucial role in the susceptibility and/or resistance to many human diseases, but little is known about Tibetan macaques. To gain an insight into the MHC background and to facilitate the experimental use of Tibetan macaques, the second exon of Mhc-DQB1 gene was sequenced in a cohort of wild Tibetan macaques living in the Sichuan province of China. A total of 23 MhcMath-DQB1 alleles were identified for the first time, illustrating a marked allelic polymorphism at the DQB1 locus for these macaques. Most of the sequences (74%) observed in this study belong to DQB1*06 (9 alleles) and DQB1*18 (8 alleles) lineages, and the rest (26%) belong to DQB1*15 (3 alleles) and DQB1*17 (3 alleles) lineages. The most frequent alleles detected among these macaques were MhcMath-DQB1*15:02:02 (17.9%), followed by Math-DQB1*06:06, 17:03 and 18:01, which were detected in 9 (16.1%) of the monkeys, respectively. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the peptide-binding region, suggesting balancing selection for maintaining polymorphisms at the MHC class II DQB1 locus. Phylogenetic analyses confirms the trans-species model of evolution of the Mhc-DQB1 genes in non-human primates, and in particular, the extensive allele sharing is observed between Tibetan and other macaque species., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Self-injurious behavior (SIB) is a complex condition that exhibits a spectrum of abnormal neuropsychological and locomotor behaviors. Mechanisms for neuropathogenesis could include irregular immune activation, host soluble factors, and astrocyte dysfunction., Methods: We examined the role of astrocytes as modulators of immune function in macaques with SIB. We measured changes in astrocyte morphology and function. Paraffin sections of frontal cortices from rhesus macaques identified with SIB were stained for glial fibrillary acidic protein (GFAP) and Toll-like receptor 2 (TLR2). Morphologic features of astrocytes were determined using computer-assisted camera lucida., Results: There was atrophy of white matter astrocyte cell bodies, decreased arbor length in both white and gray matter astrocytes, and decreased bifurcations and tips on astrocytes in animals with SIB. This was combined with a five-fold increase in the proportion of astrocytes immunopositive for TLR2., Conclusions: These results provide direct evidence that SIB induces immune activation of astrocytes concomitant with quantifiably different morphology.

Lassa virus (LASV) is a significant human pathogen that is endemic to several countries in West Africa. Infection with LASV leads to the development of hemorrhagic fever in a significant number of cases, and it is estimated that thousands die each year from the disease. Little is known about the complex immune mechanisms governing the response to LASV or the genetic determinants of susceptibility and resistance to infection. In the study presented here, we have used a whole-genome, microarray-based approach to determine the temporal host response in the peripheral blood mononuclear cells (PBMCs) of non-human primates (NHP) following aerosol exposure to LASV. Sequential sampling over the entire disease course showed that there are strong transcriptional changes of the immune response to LASV exposure, including the early induction of interferon-responsive genes and Toll-like receptor signaling pathways. However, this increase in early innate responses was coupled with a lack of pro-inflammatory cytokine response in LASV exposed NHPs. There was a distinct lack of cytokines such as IL1β and IL23α, while immunosuppressive cytokines such as IL27 and IL6 were upregulated. Comparison of IRF/STAT1-stimulated gene expression with the viral load in LASV exposed NHPs suggests that mRNA expression significantly precedes viremia, and thus might be used for early diagnostics of the disease. Our results provide a transcriptomic survey of the circulating immune response to hemorrhagic LASV exposure and provide a foundation for biomarker identification to allow clinical diagnosis of LASV infection through analysis of the host response.

The envelope glycoproteins (Env) represent a critical component of a successful antibody-mediated human immunodeficiency virus type 1 (HIV-1) vaccine. However, immunization with soluble Env was reported to induce short-lived antibody responses, suggesting that Env has unusual immunogenic properties. Here, we directly compared the magnitude and durability of B-cell responses induced by HIV-1 Env and an unrelated soluble viral protein, influenza virus hemagglutinin (HA), in simultaneously inoculated macaques. We demonstrate robust peak responses followed by rapid contraction of circulating antibody and memory B cells for both antigens, suggesting that short-lived responses are not unique to HIV-1 Env but may be a common feature of soluble protein vaccines.

Major histocompatibility complex (MHC) gene products control the repertoire of T cell responses that an individual may create against pathogens and foreign tissues. This text will review the current understanding of MHC genetics in nonhuman primates, with a focus on Mauritian-origin cynomolgus macaques (Macaca fascicularis) and Indian-origin rhesus macaques (Macaca mulatta). These closely related macaque species provide important experimental models for studies of infectious disease pathogenesis, vaccine development, and transplantation research. Recent advances resulting from the application of several cost effective, high-throughput approaches, with deep sequencing technologies have revolutionized our ability to perform MHC genotyping of large macaque cohorts. Pyrosequencing of cDNA amplicons with a Roche/454 GS Junior instrument, provides excellent resolution of MHC class I allelic variants with semi-quantitative estimates of relative levels of transcript abundance. Introduction of the Illumina MiSeq platform significantly increased the sample throughput, since the sample loading workflow is considerably less labor intensive, and each instrument run yields approximately 100-fold more sequence data. Extension of these sequencing methods from cDNA to genomic DNA amplicons further streamlines the experimental workflow and opened opportunities for retrospective MHC genotyping of banked DNA samples. To facilitate the reporting of MHC genotypes, and comparisons between groups of macaques, this text also introduces an intuitive series of abbreviated rhesus MHC haplotype designations based on a major Mamu-A or Mamu-B transcript characteristic for ancestral allele combinations. The authors believe that the use of MHC-defined macaques promises to improve the reproducibility, and predictability of results from pre-clinical studies for translation to humans.

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as an important animal model for infectious disease and transplantation research. Extensive characterization of their major histocompatibility complex (MHC) polymorphism regions therefore becomes urgently required. In this study, we identified 41 MHC class I A nucleotide sequences in 34 unrelated cynomolgus macaques of Vietnamese origin farmed in Southern China, including eight novel Mafa-A sequences. We found two sequences with perfect identity and six sequences with close similarity to previously defined MHC class I alleles from other populations, especially from Indonesian-origin macaques. We also found three Vietnamese-origin cynomolgus macaque MHC class I sequences for which the predicted protein sequences identical throughout their B and F binding pockets to Mamu-A1*001:01 and Mamu-A3*13:03, respectively. This is important because Mamu-A1*001:01 and Mamu-A3*13:03 are associated with longer survival and lower set-point viral load in simian immunodeficiency virus (SIV)-infected rhesus monkeys. These findings have implications for the evolutionary history of Vietnamese-origin cynomolgus macaque as well as for the use of this model in SIV/SHIV (a virus combining parts of the HIV and SIV genomes) research., (© 2012 John Wiley & Sons A/S.)

This study systematically validated two quantitative enzyme-linked immunosorbent assays (ELISAs) for determining Yersinia pestis anti-F1 or anti-V IgG concentration in cynomolgus macaque sera. The results demonstrated that these ELISAs are reliable, reproducible, and suitable for their intended use to measure both anti-F1 and anti-V IgG in monkey sera following vaccination with a heterologous recombinant fusion F1-V protein (rF1-V). Statistical analysis demonstrated assay precision, accuracy, specificity, linearity/dilutional linearity, and robustness for both assays. The quantitative ranges of standard curves were defined as 40-700 ng/mLfor both anti-F1 and anti-V IgG. Either serological assay could be used to determine potency of F1/V antigen-based vaccines in surrogate clinical studies or to define correlates of protective immunity against plague under the Food and Drug Administration's (FDA) two-animal rule.