Long-primed germinal centres with enduring affinity maturation and clonal migration.

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 (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Duration of antiretroviral therapy impacts the degree of residual SIV infection in the gut in long-term non-progressing Chinese rhesus macaques.

The gut is a major reservoir in HIV-infected individuals on antiretroviral therapy (ART) and in long-term non-progressors (LTNPs). Whether ART reduces gut infection and reservoirs in LTNPs is unknown. Herein, SIV-infected LTNP Rhesus macaques were treated with short- or long-term ART, and SIV envelope gp120 sequences obtained from single genome amplification were analyzed before and after ART in peripheral blood and the intestine. Although ART does not eliminate SIV in these LTNPs, a longer ART period dramatically reduces SIV infection in the gut. This study highlights the importance of long-term ART in LTNPs to minimize gut infection and prolong remission.

Comparison of fine-needle aspiration techniques.

BACKGROUND: Fine-needle aspiration (FNA) has been reported since 1912 beginning with the use of trocars and other specialized instruments that were impractical. Since then, FNA has proven to be a successful alternative technique to excisional biopsy for some assays despite a few limitations. METHODS: In this study, we compared four different techniques for FNA in rhesus macaques by evaluating total live cells recovered and cell viability using a standard 6 mL syringe and 1.5-inch 22-gauge needle. RESULTS: Technique B which was the only technique in which the needle was removed from the syringe after collection of the sample to allow forced air through the needle to expel the contents into media followed by flushing of the syringe and needle resulted in the highest total cell count and second highest cell viability in recovered cells. CONCLUSION: Based on our results, Technique B appears to be the superior method.

Chronic binge alcohol and ovariectomy-mediated impaired insulin responsiveness in SIV-infected female rhesus macaques.

Aging people living with HIV (PLWH), especially postmenopausal women may be at higher risk of comorbidities associated with HIV, antiretroviral therapy (ART), hypogonadism, and at-risk alcohol use. Our studies in simian immunodeficiency virus (SIV)-infected male macaques demonstrated that chronic binge alcohol (CBA) reduced acute insulin response to glucose (AIRG), and at-risk alcohol use decreased HOMA-ß in PLWH. The objective of this study was to examine the impact of ovariectomy (OVX) on glucose-insulin dynamics and integrity of pancreatic endocrine function in CBA/SIV-infected female macaques. Female macaques were administered CBA (12-15 g/kg/wk) or isovolumetric water (VEH) intragastrically. Three months after initiation of CBA/VEH administration, all macaques were infected with SIVmac251, and initiated on antiretroviral therapy (ART) 2.5 mo postinfection. After 1 mo of ART, macaques were randomized to OVX or sham surgeries (n = 7 or 8/group), and euthanized 8 mo post-OVX (study endpoint). Frequently sampled intravenous glucose tolerance tests (FSIVGTT) were performed at selected time points. Pancreatic gene expression and islet morphology were determined at study endpoint. There was a main effect of CBA to decrease AIRG at Pre-SIV and study endpoint. There were no statistically significant OVX effects on AIRG (P = 0.06). CBA and OVX decreased the expression of pancreatic markers of insulin docking and release. OVX increased endoplasmic stress markers. CBA but not OVX impaired glucose-insulin expression dynamics in SIV-infected female macaques. Both CBA and OVX altered integrity of pancreatic endocrine function. These findings suggest increased vulnerability of PLWH to overt metabolic dysfunction that may be exacerbated by alcohol use and ovarian hormone loss.

Chronic binge alcohol administration impairs glucose-insulin dynamics and decreases adiponectin in asymptomatic simian immunodeficiency virus-infected macaques.

Alcohol use disorders (AUDs) frequently exist among persons living with HIV/AIDS. Chronic alcohol consumption, HIV infection, and antiretroviral therapy (ART) are independently associated with impairments in glucose-insulin dynamics. Previous studies from our laboratory have shown that chronic binge alcohol (CBA) administration decreases body mass index, attenuates weight gain, and accentuates skeletal muscle wasting at end-stage disease in non-ART-treated simian immunodeficiency virus (SIV)-infected male rhesus macaques. The aim of this study was to investigate whether CBA and ART alone or in combination alter body composition or glucose-insulin dynamics in SIV-infected male rhesus macaques during the asymptomatic phase of SIV infection. Daily CBA or sucrose (SUC) administration was initiated 3 mo before intrarectal SIV inoculation and continued until the study end point at 11 mo post-SIV infection. ART or placebo was initiated 2.5 mo after SIV infection and continued until study end point. Four treatment groups (SUC/SIV ± ART and CBA/SIV ± ART) were studied. CBA/SIV macaques had significantly decreased circulating adiponectin and resistin levels relative to SUC/SIV macaques and reduced disposition index and acute insulin response to glucose, insulin, and C-peptide release during frequently sampled intravenous glucose tolerance test, irrespective of ART status. No statistically significant differences were observed in homeostatic model assessment-insulin resistance values, body weight, total body fat, abdominal fat, or total lean mass or bone health among the four groups. These findings demonstrate CBA-mediated impairments in glucose-insulin dynamics and adipokine profile in asymptomatic SIV-infected macaques, irrespective of ART.

Ethanol impairs mucosal immunity against Streptococcus pneumoniae infection by disrupting interleukin 17 gene expression.

Acute ethanol intoxication suppresses the host immune responses against Streptococcus pneumoniae. As interleukin 17 (IL-17) is a critical cytokine in host defense against extracellular pathogens, including S. pneumoniae, we hypothesized that ethanol impairs mucosal immunity against this pathogen by disrupting IL-17 production or IL-17 receptor (IL-17R) signaling. A chronic ethanol feeding model in simian immunodeficiency virus (SIV)-infected rhesus macaques and acute ethanol intoxication in a murine model were used. Transcriptome analysis of bronchial brushes in the nonhuman primate model showed downregulation of the expression of IL-17-regulated chemokines in ethanol-fed animals, a finding also replicated in the murine model. Surprisingly, recombinant CXCL1 and CXCL5 but not IL-17 or IL-23 plus IL-1ß rescued bacterial burden in the ethanol group to control levels. Taken together, the results of this study suggest that ethanol impairs IL-17-mediated chemokine production in the lung. Thus, exogenous luminal restoration of IL-17-related chemokines, CXCL1 and CXCL5, improves host defenses against S. pneumoniae.

Non-human primate model of long-COVID identifies immune associates of hyperglycemia.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.

The Impact of SIV-Induced Immunodeficiency on SARS-CoV-2 Disease, Viral Dynamics, and Antiviral Immune Response in a Nonhuman Primate Model of Coinfection.

The effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication, and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.

Cytomegalovirus vaccine vector-induced effector memory CD4 + T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge.

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.