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Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (B

While T cell immunity is an important component of the immune response to Zika virus (ZIKV) infection generally, the efficacy of these responses during pregnancy remains unknown. Here, we tested the capacity of CD8 lymphocytes to protect from secondary challenge in four macaques, two of which were depleted of CD8+ cells prior to rechallenge with a heterologous ZIKV isolate. The initial challenge during pregnancy produced transcriptional signatures suggesting complex patterns of immune modulation as well as neutralizing antibodies that persisted until rechallenge, which all animals efficiently controlled, demonstrating that the primary infection conferred adequate protection. The secondary challenge promoted activation of innate and adaptive immune cells, possibly suggesting a brief period of infection prior to clearance. These data confirm that ZIKV infection during pregnancy induces sufficient immunity to protect from a secondary challenge and suggest that this protection is not dependent on CD8 T cells., Highlights • Zika infection during pregnancy provides protection from secondary infection. • CD8 T cell responses are not needed for protection from secondary infection. • Zika infection during pregnancy results in immunomodulatory transcriptional signatures but little IFN response.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

SummaryGerminal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics., Author summary The COVID-19 pandemic has claimed the lives of millions in a span of less than two years. Despite the development of several highly effective vaccines, many millions remain unvaccinated, and several highly transmissible variants have emerged, clearly suggesting the need for new approaches to treat those that become severely ill. The development of new drugs will rely on having animal models that reproduce the most severe disease seen in humans. To date, nonhuman primate models have not exhibited this severe disease. In this study we tested whether pigtail macaques (PTM) might exhibit such severe disease, based on previous work showing this species is prone to more rapid and severe disease when infected with other viruses.

The COVID-19 pandemic resulted from global infection by the SARS-CoV-2 coronavirus and rapidly emerged as an urgent health issue requiring effective treatments. To initiate infection, the Spike protein of SARS-CoV-2 requires proteolytic processing mediated by host proteases. Among the host proteases proposed to carry out this activation is the cysteine protease cathepsin L. Inhibiting cathepsin L has been proposed as a therapeutic strategy for treating COVID-19. SLV213 (K777) is an orally administered small molecule protease inhibitor that exhibits in vitro activity against a range of viruses, including SARS-CoV-2. To confirm efficacy in vivo, K777 was evaluated in an African green monkey (AGM) model of COVID-19. A pilot experiment was designed to test K777 in a prophylactic setting, animals were pre-treated with 100mg/kg K777 (N=4) or vehicle (N=2) before inoculation with SARS-CoV-2. Initial data demonstrated that K777 treatment reduced pulmonary pathology compared to vehicle-treated animals. A second study was designed to test activity in a therapeutic setting, with K777 treatment (33 mg/kg or 100 mg/kg) initiated 8 hours after exposure to the virus. In both experiments, animals received K777 daily via oral gavage for 7 days. Vehicle-treated animals exhibited higher lung weights, pleuritis, and diffuse alveolar damage. In contrast, lung pathology was reduced in K777-treated monkeys, and histopathological analyses confirmed the lack of diffuse alveolar damage. Antiviral effects were further demonstrated by quantitative reductions in viral load of samples collected from upper and lower airways. These preclinical data support the potential for early SLV213 treatment in COVID-19 patients to prevent severe lung pathology and disease progression.

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

SUMMARYThe HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion that encodes amino acids 734-736 (ΔQTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naïve PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

Summary Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCVs) from nonhuman primates are transmitted through oral secretions, penetrate the mucosal epithelium, and establish persistent infection in B cells. To determine whether neutralizing antibodies against epithelial or B cell infection could block oral transmission and persistent LCV infection, we use rhesus macaques, the most accurate animal model for EBV infection by faithfully reproducing acute and persistent infection in humans. Naive animals are infused with monoclonal antibodies neutralizing epithelial cell infection or B cell infection and then challenged orally with recombinant rhesus LCV. Our data show that high-titer B cell-neutralizing antibodies alone, but not epithelial cell-neutralizing antibodies, can provide complete protection of rhesus macaques from oral LCV challenge, but not in all hosts. Thus, neutralizing antibodies against B cell infection are important targets for EBV vaccine development, but they may not be sufficient., Graphical abstract, Highlights mAb infusion leads to high neutralizing titers in nonhuman primates Protection of epithelial cells does not protect from lymphocryptovirus challenge Neutralization of B cell infection alone provides partial protection in macaques, Epstein-Barr virus is associated with a variety of diseases and causes a burden for healthcare and the economy. Thus, a vaccine is urgently needed. Mühe et al. provide insights into how protection from natural infection could be achieved and which viral antigens are pivotal for successful vaccine development.

Nonhuman primates (NHPs) in research institutions may be housed in a variety of social settings, such as group housing, pair housing or single housing based on the needs of studies. Furthermore, housing may change over the course of studies. The effects of housing and changes in housing on cell activation and vaccine mediated immune responses are not well documented. We hypothesized that animals moved indoors from group to single housing (GH-SH) would experience more stress than those separated from groups into pair housing (GH-PH), or those placed briefly into pair housing and separated 5 weeks later into single housing (GH-PH-SH). We also compared the effects of separation from group to pair housing with the separation from pair to single housing. Eighteen male rhesus macaques were followed over the course of changes in housing condition over 10–14 weeks, as well as prior to and after primary vaccination with a commercially available measles vaccine. We identified two phenotypic biomarkers, namely total CD8 population and proliferating B cells, that differed significantly across treatment groups over time. At 10 weeks post-separation, levels of proliferating B cells were higher in GH-SH subjects compared to GH-PH subjects, and in the latter, levels were lower at 10 weeks than prior to removal from group housing. At 2 weeks post-separation from group to single housing, the frequency of CD8+ T cells was higher in GH-SH subjects compared to one week post separation from pair into single housing in the GH-PH-SH subjects. Comparing the same elapsed time since the most recent separation activated CD20 populations were persistently higher in the GH-SH animals than the GH-PH-SH animals. Housing configuration did not influence vaccine-mediated responses. Overall, our study found benefits of pair housing over single housing, suggesting that perturbations in immune function will be more severe following separation from group to single housing than from pair to single housing, and supporting the use of short-duration pair housing even when animals must subsequently be separated. These findings are useful for planning the housing configurations of research NHPs used for vaccine studies and other studies where immune response is being assessed.

Summary We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.

While T cell immunity is an important component of the immune response to Zika virus (ZIKV) infection generally, the efficacy of these responses during pregnancy remains unknown. Here, we tested the capacity of CD8 lymphocytes to protect from secondary challenge in four macaques, two of which were depleted of CD8+ cells prior to rechallenge with a heterologous ZIKV isolate. The initial challenge during pregnancy produced transcriptional signatures suggesting complex patterns of immune modulation, but all animals efficiently controlled the rechallenge virus, implying that the primary infection conferred adequate protection. The secondary challenge promoted humoral responses and activation of innate and adaptive immune cells, suggesting a brief period of infection prior to clearance. These data confirm that ZIKV infection during pregnancy induces sufficient immunity to protect from a secondary challenge and suggest that this protection is not solely dependent on CD8 T cells but entails multiple arms of the immune system.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734–736 (ΔQTH) that overlaps therevandtatopen reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infectionin vivo.

A reduction in the burden of schistosomiasis is potentially achievable by integrating a schistosomiasis vaccine with current control measures. Here, we determine parasite-specific in vitro responses of B, T, and NK cells from naive uninfected rhesus macaques to Schistosoma mansoni (Sm) egg (SmEA) and worm antigen (SmWA) preparations isolated from infected baboons. Pronounced B cell responses to SmEA and NK cell responses to both SmEA and SmWA were observed. High levels of IL-2 and IL-21 responses against Sm antigens were observed in T and non-T cells of lymph nodes (LNs) and gut lamina propria-derived lymphocytes (LPLs). Data analysis showed multifunctionality of LN-derived CD4+ , CD8+ , and CD4+ CD8+ double positive T cells against either SmWA or SmWA+SmEA antigen preparations. Distinct SmEA-specific multifunctional responses were observed in gut LPLs, suggesting simultaneous responses against egg antigens. These data provide insight into the immune effectors involved in schistosome responses by rhesus macaques.

Zika virus (ZIKV) will remain a public health threat until effective vaccines and therapeutics are made available in the hardest hit areas of the world. Recent data in a nonhuman primate model showed that infants postnatally infected with ZIKV were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. We addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding, possibly non-neutralizing antibodies remained detectable until viral infection at 5 months of age. Post-infection acute serum viremia was substantially reduced relative to adults infected with the same dose of the same stock of a Brazilian isolate of ZIKV (n=11 pregnant females) and another stock of the same isolate (n=4 males and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection, suggesting reduced viral burden relative to adults and published data from infants. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

CD4 + follicular T helper (Tfh) cells play a prominent role in humoral immune responses, but the mechanisms of their accumulation and infection in AIDS remain unclear. Here we found that germinal center (GC) Tfh cells, defined here as CXCR5 + PD-1 HIGH CD4 + T cells, do not express the HIV coreceptor CCR5 yet serve as a latent reservoir in GCs. With disease progression, an expansion of GC Tfh cells is accompanied by increases in dysfunctional CD8 + T cells. In contrast, Tfh precursor (CXCR5 − CD4 + T) cells in lymph nodes do express CCR5 and differentiate into GC Tfh cells following interleukin-6 (IL-6) and IL-21 stimulation, and viral DNA is detectable in fully differentiated GC Tfh cells ex vivo . This suggests that SIV-infected GC Tfh cells may be derived from Tfh precursor cell subsets that become infected in marginal zones and then migrate into GCs as fully mature GC Tfh cells that serve as persistent virus reservoirs. These findings suggest that viral persistence in lymph nodes drives compensatory differentiation, aberrant accumulation, and latent infection of GC Tfh cells, resulting in marked impairment of humoral immune responses. IMPORTANCE Generation of antibodies that can effectively eliminate viruses requires interactions of B cells with highly specialized T cells in GCs of lymphoid tissues called follicular T helper cells. Here we show that in simian immunodeficiency virus infection, these cells are initially infected in a precursor stage that leads to alterations in their homing, accumulation, and function that may be responsible for the inability of human immunodeficiency virus-infected patients to generate effective antibody responses.

Zika virus (ZIKV) infection of pregnant women is associated with pathologic complications of fetal development. Here, we infect pregnant rhesus macaques (Macaca mulatta) with a minimally passaged ZIKV isolate from Rio de Janeiro, where a high rate of fetal development complications was observed. The infection of pregnant macaques with this virus results in maternal viremia, virus crossing into the amniotic fluid (AF), and in utero fetal deaths. We also treated three additional ZIKV-infected pregnant macaques with a cocktail of ZIKV-neutralizing human monoclonal antibodies (nmAbs) at peak viremia. While the nmAbs can be effective in clearing the virus from the maternal sera of treated monkeys, it is not sufficient to clear ZIKV from AF. Our report suggests that ZIKV from Brazil causes fetal demise in non-human primates (NHPs) without additional mutations or confounding co-factors. Treatment with a neutralizing anti-ZIKV nmAb cocktail is insufficient to fully stop vertical transmission., Zika virus (ZIKV) infection in pregnant women has been associated with fetal developmental defects. Here, the authors show that a Brazilian ZIKV isolate causes fetal demise in non-human primates and that antibody treatment at time of peak viremia is insufficient to clear ZIKV replication from amniotic fluid.

Vertical HIV-1 transmission via breastfeeding is the predominant contributor to pediatric infections that are ongoing in this era of highly effective antiretroviral therapy (ART). Remarkably, only ~10% of infants chronically exposed to the virus via breastfeeding from untreated HIV-infected mothers become infected, suggesting the presence of naturally protective factors in breast milk. HIV-specific maternal antibodies are obvious candidates as potential contributors to this protection. This study assessed the protective capacity of common HIV envelope-specific non-broadly neutralizing antibodies isolated from breast milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. Prior to oral SHIV challenge, infant RMs were i.v. infused with either a single weakly-neutralizing monoclonal antibody (mAb), a tri-mAb cocktail with neutralizing and ADCC functionalities, or an anti-influenza HA control mAb. Of these groups, the fewest tri-mAb-treated infants developed plasma viremia (2/6, 3/6, and 6/8 animals viremic in tri-mAb, single-mAb, and control mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer transmitted/founder SHIV variants in plasma and decreased peripheral CD4+ T cell proviral loads at 8 week post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single-mAb-, but not tri-mAb-treated animals. Taken together, these results support the potential viability of maternal or infant vaccine strategies that elicit non-broadly neutralizing antibodies to prevent vertical transmission of HIV through breastfeeding.

HIV replication and the cellular micro-RNA (miRNA) machinery interconnect at several posttranscriptional levels. To understand their regulatory role in the intestine, a major site of HIV/SIV replication, dissemination, and CD4+ T cell depletion, we profiled miRNA expression in colon following SIV infection (10 acute SIV, 5 uninfected). Nine (four up and five down) miRNAs showed statistically significant differential expression. Most notably, miR-190b expression showed high statistical significance (adjusted p = 0.0032), the greatest fold change, and was markedly elevated in colon and jejunum throughout SIV infection. In addition, miR-190b upregulation was detected before peak viral replication and the nadir of CD4+ T cell depletion predominantly in lamina propria leukocytes. Interestingly non–SIV-infected macaques with diarrhea and colitis failed to upregulate miR-190b, suggesting that its upregulation was neither inflammation nor immune-activation driven. SIV infection of in vitro–cultured CD4+ T cells and primary intestinal macrophages conclusively identified miR-190b upregulation to be driven in response to viral replication. Further miR-190b expression levels in colon and jejunum positively correlated with tissue viral loads. In contrast, mRNA expression of myotubularin-related protein 6 (MTMR6), a negative regulator of CD4+ T cell activation/proliferation, significantly decreased in SIV-infected macrophages. Luciferase reporter assays confirmed MTMR6 as a direct miR-190b target. To our knowledge, this is the first report, which describes dysregulated miRNA expression in the intestine, that identifies a potentially significant role for miR-190b in HIV/SIV pathogenesis. More importantly, miR-190b–mediated MTMR6 downregulation suggests an important mechanism that could keep infected cells in an activated state, thereby promoting viral replication. In the future, the mechanisms driving miR-190b upregulation including other cellular processes it regulates in SIV-infected cells need determination.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement—but not remission—of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm—by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-γ and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea—all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site. Of the seven mutations that differentiate iMac239 from wild-type SIVmac239, a single change (D178G) in the V1/V2 region was sufficient to confer CD4 independence in cell-cell fusion assays, although other mutations were required for replication competence. Like other CD4-independent viruses, iMac239 was highly neutralization sensitive, although mutations were identified that could confer CD4-independent infection without increasing its neutralization sensitivity. Strikingly, iMac239 retained the ability to replicate in cell lines and primary cells even when its CD4 binding site had been ablated by deletion of a highly conserved aspartic acid at position 385, which, for HIV-1, plays a critical role in CD4 binding. iMac239, with and without the D385 deletion, exhibited an expanded host range in primary rhesus peripheral blood mononuclear cells that included CCR5 + CD8 + T cells. As the first non-CD4-tropic SIV, iMac239-ΔD385 will afford the opportunity to directly assess the in vivo role of CD4 targeting on pathogenesis and host immune responses. IMPORTANCE CD4 tropism is an invariant feature of primate lentiviruses and likely plays a key role in pathogenesis by focusing viral infection onto cells that mediate adaptive immune responses and in protecting virions attached to cells from neutralizing antibodies. Although CD4-independent viruses are well described for HIV and SIV, these viruses characteristically retain their CD4 binding site and can engage CD4 if available. We derived a novel CD4-independent, CCR5-tropic variant of the pathogenic molecular clone SIVmac239, termed iMac239. The genetic determinants of iMac239's CD4 independence provide new insights into mechanisms that underlie this phenotype. This virus remained replication competent even after its CD4 binding site had been ablated by mutagenesis. As the first truly non-CD4-tropic SIV, lacking the capacity to interact with CD4, iMac239 will provide the unique opportunity to evaluate SIV pathogenesis and host immune responses in the absence of the immunomodulatory effects of CD4 + T cell targeting and infection.

N. A. Renner, R. K. Redmann, T. Moroney-Rasmussen, H. A. Sansing, P. P. Aye, P. J. Didier, A. A. Lackner and A. G. MacLean (2012) Neuropathology and Applied Neurobiology38, 162–174 S100β as a novel and accessible indicator for the presence of monocyte-driven encephalitis in AIDS Aims: The pathogenesis of human/simian immunodeficiency virus encephalitis (HIVE/SIVE) remains incompletely understood, but is associated with alterations in the blood–brain barrier. At present, it is not possible to easily determine if an individual has HIVE/SIVE before post mortem examination. Methods: We have examined serum levels of the astroglial protein S100β in SIV-infected macaques and show that it can be used to determine which animals have SIVE. We also checked for correlations with inflammatory markers such as CCL2/MCP-1, IL-6 and C-reactive protein. Results: We found that increased S100β protein in serum correlated with decreased expression of the tight junction protein zonula occludens-1 on brain microvessels. Furthermore, the decrease in zonula occludens-1 expression was spatially related to SIVE lesions and perivascular deposition of plasma fibrinogen. There was no correlation between encephalitis and plasma levels of IL-6, MCP-1/CCL2 or C-reactive protein. Conclusions: Together, these data indicate that SIVE lesions are associated with vascular leakage that can be determined by S100β protein in the periphery. The ability to simply monitor the presence of SIVE will greatly facilitate studies of the neuropathogenesis of AIDS.

Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.

Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc(2)6020 (DeltalysA DeltapanCD) and mc(2)6030 (DeltaRD1 DeltapanCD) in cynomolgus macaques. In murine models, mc(2)6020 is rapidly cleared while mc(2)6030 persists. Both mc(2)6020 and mc(2)6030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc(2)6020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc(2)6030 vaccinates, but survival did not differ among the groups.

Background Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. Methods We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. Results Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. Conclusions These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.

Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model ofMycobacterium tuberculosisinfection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonaryM. tuberculosisinfection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated withM. tuberculosisinfection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergotrans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses toM. tuberculosisinfection.

Gastrointestinal disease and inflammation are common sequelae of human and simian immunodeficiency virus (SIV) infection. Nevertheless, the molecular mechanisms that lead to gastrointestinal dysfunction remain unclear. We investigated regulation of the interleukin (IL)-6-JAK-STAT3 pathway in jejunum and colon, collected at necropsy, from 10 SIV-infected macaques with diarrhea (group 1), 10 non-SIV-infected macaques with diarrhea (group 2), and 7 control uninfected macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more frequent and severe lesions in the jejunum. A significant increase in IL-6 and SOCS-3 gene expression along with constitutive STAT3 activation was observed in the colon of all group 1 and 2 macaques and in the jejunum of only group 1 macaques compared to controls. Further, in colon, histopathology severity scores correlated significantly with IL-6 (groups 1 and 2) and SOCS-3 (group 2) gene expression. In jejunum, a similar correlation was observed only in group 1 animals. Phosphorylated STAT3 (p-STAT3) was localized to lymphocytes (CD3 + ) and macrophages (CD68 + ), with fewer CD3 + lymphocytes expressing p-STAT3 in group 1 macaques. Despite high SOCS-3 expression, STAT3 remained constitutively active, providing a possible explanation for persistent intestinal inflammation and immune activation that may favor viral replication and disease pro-gression.

It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks, mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells (PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells. To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry were used to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected by RT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the published SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocal microscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examined by multicolor flow cytometry, SERT bright cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lymphocytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would be suitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.

Cryptosporidiosis, caused by the protozoan parasiteCryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease.Cryptosporidiuminfection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl−) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl−secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and afterCryptosporidium parvuminfection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl−secretion and glucose malabsorption, was studied using tissues derived from macaques infected withC. parvumby the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples followingC. parvuminfection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques ( Macaca mulatta ). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4 + T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques ( Macaca nemestrina ), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4 + T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of + T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4 + T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8 + cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

The difficulty in developing an effective vaccine to contain the HIV/AIDS epidemic coupled with the fact that primary HIV-1 infection typically occurs via mucosal sites has increased emphasis on vaccine approaches that protect at mucosal surfaces. In this study we employed HIV and simian-HIV (SHIV)-derived T helper (Th) and cytotoxic T lymphocyte (CTL) single epitopes incorporated into immuno-stimulating complexes (ISCOM) as a candidate immunogens. Immunized rhesus macaques (Macaca mulatta) were challenged with CCR5-tropic SHIV(SF162p4). On the day of challenge, low levels of virus-neutralizing antibodies (Ab) and CTLs were detected in ISCOM-immunized macaques. Greater than 10(5) viral RNA copies per ml of plasma in 2/5 immunized and 3/4 control macaques were detected within 3 weeks post-challenge. Depletion of CD4+ T cells from gut-associated lymphoid tissues (GALT) was observed by post-challenge day (PCD) 14 in all macaques regardless immunization. Nonetheless, lower viral loads and relatively better preservation of peripheral CD4+ T cells following the SHIV infection was observed in ISCOM-immunized macaques. We predict that if coadministered with additional epitopes and/or more efficacious mucosal delivery system or route, HIV/SIV-derived peptide vaccines may have potential to elicit heterologous protection.

Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-α)-, and IL-1-α-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-α-producing cells were T lymphocytes (CD3+), while the TNF-α-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P< 0.001) than that seen in the healthy control animal, while TNF-α- and IL-1-α-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.

SIVmac239/316 is a molecular clone derived from SIVmac239 that differs from the parental virus by nine amino acids in env. This virus, unlike the parental SIVmac239, is able to replicate well in alveolar macrophages in culture. We have not however, observed macrophage-associated inflammatory disease in any animal infected with SIVmac239/316. Therefore, we sought to examine the cell tropism of this virus in vivo in multiple tissues using in situ hybridization combined with immunohistochemistry and multilabel confocal microscopy for viral nucleic acid and multiple cell-type-specific markers for macrophages and T lymphocytes. Tissues examined included brain, heart, lung, lymph nodes, spleen, thymus, and small and large intestine. Matched tissues from macaques infected with the parental SIVmac239 and uninfected macaques were also examined. Many infected cells were detected in the tissues of animals infected with SIVmac239 and SIVmac239/316 although there appeared to be fewer positive cells in animals infected with SIVmac239/316. Surprisingly, in light of the cell culture observations, nearly every simian immunodeficiency virus-infected cell in animals inoculated with SIVmac239/316 was a T lymphocyte rather than a macrophage. This was true both during early infection (first 2 months) and in terminal disease. In contrast, as previously described, SIVmac239 was found in both T cells and macrophages in tissues as early as 21 days after infection. These studies indicate that during both acute and chronic SIVmac239/316 infection T lymphocytes rather than macrophages are the principal targets in vivo. These data combined with the absence of macrophage-associated lesions in SIVmac239/316-infected animals indicate that in vitro cell tropism is not predictive of in vivo tropism or disease pathogenesis.

The appearance of virus-specific CD4 + and/or CD8 + T lymphocytes in peripheral blood of captive juvenile rhesus macaques ( Macaca mulatta ) was observed following rotavirus infection. These cell-mediated immune responses were measured following experimental or natural infection after rotavirus was isolated from stool specimens of asymptomatic animals. The virus isolated was a new strain of simian rotavirus that we named TUCH (for Tulane University and Cincinnati Children's Hospital). Restimulation of peripheral T lymphocytes by inactivated double- or triple-layered TUCH rotavirus particles containing either VP6 or VP4 and VP7 on their respective surfaces resulted in increased quantities of interleukin-6 (IL-6) and IL-12 in cell culture supernatants. Recall responses to rotavirus by CD4 + and CD8 + T lymphocytes were associated with accumulation of intracellular IL-6 and gamma interferon. Antigen presentation of TUCH rotavirus to lymphocytes was mediated via differentiated cultures of monocyte-derived dendritic (HLA-DR + ) cells. This is the first report demonstrating cell-mediated immune responses to rotavirus in nonhuman primates. Further exploration of rhesus macaques in vaccine trials with human rotavirus vaccine candidates is the major objective of future studies.

Celiac disease (CD) affects approximately 1% of the general population while an estimated additional 6% suffers from a recently characterized, rapidly emerging, similar disease, referred to as non-celiac gluten sensitivity (NCGS). The only effective treatment of CD and NCGS requires removal of gluten sources from the diet. Since required adherence to a gluten-free diet (GFD) is difficult to accomplish, efforts to develop alternative treatments have been intensifying in recent years. In this study, the non-human primate model of CD/NCGS, e.g., gluten-sensitive rhesus macaque, was utilized with the objective to evaluate the treatment potential of reduced gluten cereals using a reduced gluten (RG, 1% of normal gluten) barley mutant as a model. Conventional and RG barleys were used for the formulation of experimental chows and fed to gluten-sensitive (GS) and control macaques to determine if RG barley causes a remission of dietary gluten-induced clinical and immune responses in GS macaques. The impacts of the RG barley diet were compared with the impacts of the conventional barley-containing chow and the GFD. Although remission of the anti-gliadin antibody (AGA) serum responses and an improvement of clinical diarrhea were noted after switching the conventional to the RG barley diet, production of inflammatory cytokines, e.g., interferon-gamma (IFN-γ), tumor necrosis factor (TNF) and interleukin-8 (IL-8) by peripheral CD4+ T helper lymphocytes, persisted during the RG chow treatment and were partially abolished only upon re-administration of the GFD. It was concluded that the RG barley diet might be used for the partial improvement of gluten-induced disease but its therapeutic value still requires upgrading—by co-administration of additional treatments.

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette–Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative–specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis.

Disruption of the conserved motif GYxxØ in the simian immunodeficiency virus (SIV) SIVmac239 envelope (Env) cytoplasmic tail resulted in a virus (ΔGY) that exhibited a high plasma peak but uniquely failed to acutely deplete mucosal CD4 + T cells. Here, we show that ΔGY containing a flanking S727P mutation that was acquired in ΔGY-infected macaques reacquired the ability to rapidly deplete CD4 + T cells in lamina propria. This suggests that the GYxxØ motif and S727P each contribute to SIV's targeting to mucosal tissues.

A hallmark of pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections is the rapid and near-complete depletion of mucosal CD4 + T lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here, we evaluate in rhesus macaques a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4 + T cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation and declining peripheral blood CD4 + T cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or central nervous system tissues even in late-stage disease. Although the ΔGY mutation persisted, novel mutations evolved, including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wild-type levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.

The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS) captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD), gluten-free diet (GFD), barley gluten-derived diet (BOMI) and reduced gluten barley-derived diet (RGB). It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p < 0.05) following the consumption of dietary gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae) were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity) of gut microbiome in GS macaques started to change (p = 0.011) towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b) were upregulated (p < 0.05) in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911), Prevotella stercorea (NR_041364) and Streptococcus luteciae (AJ297218) that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes in GS macaques will approach those of healthy individuals. Further studies are needed to elucidate the regulatory pathways of inflammatory miRNAs in intestinal mucosa of GS macaques and to correlate their expression with gut dysbiosis.

Background HIV infection causes a qualitative and quantitative loss of CD4+ T cell immunity. The institution of anti-retroviral therapy (ART) restores CD4+ T cell responses to many pathogens, but HIV-specific responses remain deficient. Similarly, therapeutic immunization with HIV antigens of chronically infected, ART treated subjects results in poor induction of HIV-specific CD4 responses. In this study, we used a macaque model of ART treatment during chronic infection to study the virologic consequences of SIV antigen stimulation in lymph nodes early after immunization. Rhesus CMV (RhCMV) seropositive, Mamu A*01 positive rhesus macaques were chronically infected with SIVmac251 and treated with ART. The immune and viral responses to SIV gag and RhCMV pp65 antigen immunization in draining lymph nodes and peripheral blood were analyzed. Animals were immunized on contralateral sides with SIV gag and RhCMV pp65 encoding plasmids, which allowed lymph nodes draining each antigen to be obtained at the same time from the same animal for direct comparison. Results We observed that both SIV and RhCMV immunizations stimulated transient antigen-specific T cell responses in draining lymph nodes. The RhCMV-specific responses were potent and sustained (50 days post-immunization) in the periphery, while the SIV-specific responses were transient and extinguished quickly. The SIV antigen stimulation selectively induced transient SIV replication in draining lymph nodes. Conclusions The data are consistent with a model whereby viral replication in response to SIV antigen stimulation limits the generation of SIV antigen-specific responses and suggests a potential mechanism for the early loss and poor HIV-specific CD4+ T cell response observed in HIV-infected individuals.