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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.

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.

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.

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.

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.

The composition of gastrointestinal tract viromes has been associated with multiple diseases. Our understanding of virus communities in the GI tract is still very limited due to challenges in sampling from different GI sites. Here we defined the GI viromes of 15 rhesus macaques with chronic diarrhea. Luminal content samples from terminal ileum, proximal and distal colon were collected at necropsy while samples from the rectum were collected antemortem using a fecal loop. The composition of and ecological parameters associated with the terminal ileum virome were distinct from the colon and rectum samples; these differences were driven by bacteriophages rather than eukaryotic viruses. The six contigs that were most discriminative of the viromes were distantly related to bacteriophages from three different families. Our analysis provides support for using fecal loop sampling of the rectum as a proxy of the colonic virome in humans.

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.

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na+-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (

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.

Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory. Materials and Methods Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging. Results In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii) increased frequency of PH2AX+ (a required factor for isotype switching) B cells, iv) lower levels of Fas-L in the follicular areas, associated with lower TUNEL (cell death) positivity and v) lower accumulation of CD163+ cells (monocytes/macrophages; a surrogate of local inflammation) in proximity to follicles. The observed GC dynamics associate with earlier virus-specific antibody responses in iMacΔD compared to SIVmac239. Conclusions Our data indicate that CD4 tropism of SIV greatly affects the development of GC reactions early after infection. The iMacΔD model could provide critical information on how GC reactions can be manipulated to augment the generation of neutralizing antibodies and B cell memory.

Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles. © Springer-Verlag 2014.

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.

Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIV mac251 challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIV mac239 -GagPol, a combination of RV expressing SIV mac239 -Env and RV expressing SIV mac239 -GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At 12 weeks post-boost, all animals were challenged intravenously with 100 TCID 50 of pathogenic SIV mac251-CX . Immunized macaques in both vaccine groups had 1.3–1.6-log-fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIV mac251 neutralizing antibodies and of CD8 + T cell responses to various SIV epitopes. Moreover, vaccinated macaques better maintained peripheral memory CD4 + T cells and were able to mount a poly-functional CD8 + T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine.