FcRγ- NK Cell Induction by Specific Cytomegalovirus and Expansion by Subclinical Viral Infections in Rhesus Macaques.

"Adaptive" NK cells, characterized by FcRγ deficiency and enhanced responsiveness to Ab-bound, virus-infected cells, have been found in certain hCMV-seropositive individuals. Because humans are exposed to numerous microbes and environmental agents, specific relationships between hCMV and FcRγ-deficient NK cells (also known as g-NK cells) have been challenging to define. Here, we show that a subgroup of rhesus CMV (RhCMV)-seropositive macaques possesses FcRγ-deficient NK cells that stably persist and display a phenotype resembling human FcRγ-deficient NK cells. Moreover, these macaque NK cells resembled human FcRγ-deficient NK cells with respect to functional characteristics, including enhanced responsiveness to RhCMV-infected target in an Ab-dependent manner and hyporesponsiveness to tumor and cytokine stimulation. These cells were not detected in specific pathogen-free (SPF) macaques free of RhCMV and six other viruses; however, experimental infection of SPF animals with RhCMV strain UCD59, but not RhCMV strain 68-1 or SIV, led to induction of FcRγ-deficient NK cells. In non-SPF macaques, coinfection by RhCMV with other common viruses was associated with higher frequencies of FcRγ-deficient NK cells. These results support a causal role for specific CMV strain(s) in the induction of FcRγ-deficient NK cells and suggest that coinfection by other viruses further expands this memory-like NK cell pool.

Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction.

The implementation and access to combined antiretroviral treatment (cART) have dramatically improved the quality of life of people living with HIV (PLWH). However, some comorbidities, such as neurological disorders associated with HIV infection still represent a serious clinical challenge. Soluble factors in plasma that are associated with control of HIV replication and neurological dysfunction could serve as early biomarkers and as new therapeutic targets for this comorbidity. We used a customized antibody array for determination of blood plasma factors in 40 untreated PLWH with different levels of viremia and found sirtuin-2 (SIRT2), an NAD-dependent deacetylase, to be strongly associated with elevated viral loads and HIV provirus levels, as well as with markers of neurological damage (a-synuclein [SNCA], brain-derived neurotrophic factor [BDNF], microtubule-associated protein tau [MAPT], and neurofilament light protein [NFL]). Also, longitudinal analysis in HIV-infected individuals with immediate (n = 9) or delayed initiation (n = 10) of cART revealed that after 1 year on cART, SIRT2 plasma levels differed between both groups and correlated inversely with brain orbitofrontal cortex involution. Furthermore, targeting SIRT2 with specific small-molecule inhibitors in in vitro systems using J-LAT A2 and primary glial cells led to diminished HIV replication and virus reactivation from latency. Our data thus identify SIRT2 as a novel biomarker of uncontrolled HIV infection, with potential impact on neurological dysfunction and offers a new therapeutic target for HIV treatment and cure. IMPORTANCE Neurocognitive disorders are frequently reported in people living with HIV (PLWH) even with the introduction of combined antiretroviral treatment (cART). To identify biomarkers and potential therapeutic tools to target HIV infection in peripheral blood and in the central nervous system (CNS), plasma proteomics were applied in untreated chronic HIV-infected individuals with different levels of virus control. High plasma levels of sirtuin-2 (SIRT2), an NAD+ deacetylase, were detected in uncontrolled HIV infection and were strongly associated with plasma viral load and proviral levels. In parallel, SIRT2 levels in the peripheral blood and CNS were associated with markers of neurological damage and brain involution and were more pronounced in individuals who initiated cART later in infection. In vitro infection experiments using specific SIRT2 inhibitors suggest that specific targeting of SIRT2 could offer new therapeutic treatment options for HIV infections and their associated neurological dysfunction.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Early treatment with a combination of two potent neutralizing antibodies improves clinical outcomes and reduces virus replication and lung inflammation in SARS-CoV-2 infected macaques.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

TL1A-DR3 Plasma Levels Are Predictive of HIV-1 Disease Control, and DR3 Costimulation Boosts HIV-1-Specific T Cell Responses.

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm3, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm3, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm3) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR+CD8+ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters.

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

Neutralization of rhesus cytomegalovirus IL-10 reduces horizontal transmission and alters long-term immunity.

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.

SARS-CoV-2 surveillance for a non-human primate breeding research facility.

BACKGROUND: The emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic prompted the need for a surveillance program to determine the viral status of the California National Primate Research Center non-human primate breeding colony, both for reasons of maintaining colony health and minimizing the risk of interference in COVID-19 and other research studies. METHODS: We collected biological samples from 10% of the rhesus macaque population for systematic testing to detect SARS-CoV-2 virus by RT-PCR and host antibody response by ELISA. Testing required the development and validation of new assays and an algorithm using in laboratory-developed and commercially available reagents and protocols. RESULTS AND CONCLUSIONS: No SARS-CoV-2 RNA or antibody was detected in this study; therefore, we have proposed a modified testing algorithm for sentinel surveillance to monitor for any future transmissions. As additional reagents and controls become available, assay development and validation will continue, leading to the enhanced sensitivity, specificity, accuracy, and efficiency of testing.

Memory T Cell Proliferation before Hepatitis C Virus Therapy Predicts Antiviral Immune Responses and Treatment Success.

The contribution of the host immune system to the efficacy of new anti-hepatitis C virus (HCV) drugs is unclear. We undertook a longitudinal prospective study of 33 individuals with chronic HCV treated with combination pegylated IFN-α, ribavirin, and telaprevir/boceprevir. We characterized innate and adaptive immune cells to determine whether kinetics of the host response could predict sustained virologic response (SVR). We show that characteristics of the host immune system present before treatment were correlated with successful therapy. Augmentation of adaptive immune responses during therapy was more impressive among those achieving SVR. Most importantly, active memory T cell proliferation before therapy predicted SVR and was associated with the magnitude of the HCV-specific responses at week 12 after treatment start. After therapy initiation, the most important correlate of success was minimal monocyte activation, as predicted by previous in vitro work. In addition, subjects achieving SVR had increasing expression of the transcription factor T-bet, a driver of Th1 differentiation and cytotoxic effector cell maturation. These results show that host immune features present before treatment initiation predict SVR and eventual development of a higher frequency of functional virus-specific cells in blood. Such host characteristics may also be required for successful vaccine-mediated protection.

Subclinical Cytomegalovirus Infection Is Associated with Altered Host Immunity, Gut Microbiota, and Vaccine Responses.

Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immunocompromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.

The Effect of Early Limited Formula on Breastfeeding, Readmission, and Intestinal Microbiota: A Randomized Clinical Trial.

OBJECTIVE: To determine whether using 10 mL formula after each breastfeeding before copious maternal milk production affects breastfeeding duration, readmission, and intestinal microbiota through 1 month of age. STUDY DESIGN: In this randomized controlled trial, we enrolled 164 exclusively breastfeeding newborns, 24-72 hours old, whose weight loss was ≥75th percentile for age, and whose mothers had not yet begun mature milk production. Enrolled newborns were assigned randomly to either supplement breastfeeding with early limited formula (ELF), 10 mL of formula after each breastfeeding stopped at the onset of copious maternal milk production (intervention), or to continue exclusive breastfeeding (control). Outcomes assessed through 1 month included breastfeeding duration, readmission, and intestinal microbiota. RESULTS: At 1 week of age, 95.8% of infants receiving ELF and 93.5% of control infants were still breastfeeding (P > .5); readmission occurred for 4 (4.8%) control infants and none of the infants receiving ELF (P = .06). At 1 month of age, 86.5% of infants receiving ELF and 89.7% of control infants were still breastfeeding (P > .5); 54.6% of infants receiving ELF and 65.8% of controls were breastfeeding without formula (P = .18). ELF did not lead to decreased abundance of Lactobacillus or Bifidobacterium and was not associated with expansion of Clostridium. CONCLUSION: In this population of healthy newborns with weight loss ≥75th percentile, ELF did not interfere with breastfeeding at 1 month, breastfeeding without formula at 1 month, or intestinal microbiota. ELF may be an important therapeutic option for newborns with the potential to reduce readmission rates. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02313181.

Idiopathic Colitis in Rhesus Macaques Is Associated With Dysbiosis, Abundant Enterochromaffin Cells and Altered T-Cell Cytokine Expression.

Idiopathic chronic diarrhea (ICD) is a common ailment affecting captive rhesus macaques ( Macaca mulatta). ICD cases are characterized by diarrhea in the absence of commonly identified diarrheal pathogens and multiple recurrences even after supportive therapy. Histologically, the disease is characterized by lymphoplasmacytic colitis. We identified 35 rhesus macaques euthanized for ICD during a 7-month period and described demographic, clinical, histologic, and immunologic commonalities. We found a trend of historic Campylobacter spp. and trichomonad infections. Furthermore, rhesus macaques with ICD demonstrated loss of normal colonic adherent bacterium, identified in this study as Helicobacter macacae; increased abundance of Pentatrichomonas hominis; and increased frequency of colonic serotonin-positive enterochromaffin cells. Interestingly, colonic and ileal T-helper cells of animals with ICD manifested decreased capacity for expression of certain cytokines, in particular interleukin (IL)-4 and IL-13. These data further describe a common ailment and suggest new avenues to identify complex interactions involved in the etiology of recurring diarrhea in young rhesus macaques.

Monocyte activation by interferon α is associated with failure to achieve a sustained virologic response after treatment for hepatitis C virus infection.

BACKGROUND: Interferon α (IFN-α) and ribavirin can induce a sustained virologic response (SVR) in some but not all hepatitis C virus (HCV)-infected patients. The mechanism of effective treatment is unclear. One possibility is that IFN-α differentially improves the functional capacity of classic myeloid dendritic cells (mDCs) by altering expression of surface molecules or cytokines. Others have proposed that antigen-presenting cell activation could be paradoxically detrimental during HCV infection because of the production by monocytes of substances inhibitory or toxic to plasmacytoid dendritic cells. METHODS: We examined responses to in vitro IFN-α treatment of peripheral blood leukocyte samples from a retrospective treatment cohort of nearly 200 HCV-seropositive patients who had undergone antiviral therapy with ribavirin and pegylated IFN. We analyzed the variable responses of antigen-presenting cell subsets to drug. RESULTS: We found that patients achieving SVR were no more likely to have robust mDC activation in response to IFN-α than those who did not achieve SVR. Rather, patients achieving SVR were distinguished by restrained monocyte activation in the presence of IFN-α, a factor that was second in importance only to IL28B genotype in its association with SVR. CONCLUSIONS: These results suggest that interindividual variability in the response of monocytes to IFN-α is an important determinant of treatment success with IFN-α-based regimens.

IL-7 production in murine lymphatic endothelial cells and induction in the setting of peripheral lymphopenia.

IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.

Encapsulated Allografts Preclude Host Sensitization and Promote Ovarian Endocrine Function in Ovariectomized Young Rhesus Monkeys and Sensitized Mice.

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immunoisolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naïve ovariectomized BALB/c mice responded to the circulating gonadotropins and maintained function for 4 months, as evident by regular estrous cycles and the presence of antral follicles in the retrieved grafts. In contrast to non-encapsulated controls, repeated implantations of encapsulated mouse ovarian allografts did not sensitize naïve BALB/c mice, which was confirmed with undetectable levels of alloantibodies. Further, encapsulated allografts implanted in hosts previously sensitized by the implantation of non-encapsulated allografts restored estrous cycles similarly to our results in naïve recipients. Next, we tested the translational potential and efficiency of the immune-isolating capsule in a rhesus monkey model by implanting encapsulated ovarian auto- and allografts in young ovariectomized animals. The encapsulated ovarian grafts survived and restored basal levels of urinary estrone conjugate and pregnanediol 3-glucuronide during the 4- and 5-month observation periods. We demonstrate, for the first time, that encapsulated ovarian allografts functioned for months in young rhesus monkeys and sensitized mice, while the immunoisolating capsule prevented sensitization and protected the allograft from rejection.

Dynamics of temporal immune responses in nonhuman primates and humans immunized with COVID-19 vaccines.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

SIV clearance from neonatal macaques following transient CCR5 depletion.

Treatment of people with HIV (PWH) with antiretroviral therapy (ART) results in sustained suppression of viremia, but HIV persists indefinitely as integrated provirus in CD4-expressing cells. Intact persistent provirus, the "rebound competent viral reservoir" (RCVR), is the primary obstacle to achieving a cure. Most variants of HIV enter CD4 + T cells by binding to the chemokine receptor, CCR5. The RCVR has been successfully depleted only in a handful of PWH following cytotoxic chemotherapy and bone marrow transplantation from donors with a mutation in CCR5 . Here we show that long-term SIV remission and apparent cure can be achieved for infant macaques via targeted depletion of potential reservoir cells that express CCR5. Neonatal rhesus macaques were infected with virulent SIVmac251, then treated with ART beginning one week after infection, followed by treatment with either a CCR5/CD3-bispecific or a CD4-specific antibody, both of which depleted target cells and increased the rate of plasma viremia decrease. Upon subsequent cessation of ART, three of seven animals treated with CCR5/CD3-bispecific antibody rebounded quickly and two rebounded 3 or 6 months later. Remarkably, the other two animals remained aviremic and efforts to detect replication-competent virus were unsuccessful. Our results show that bispecific antibody treatment can achieve meaningful SIV reservoir depletion and suggest that functional HIV cure might be achievable for recently infected individuals having a restricted reservoir.

Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

The human fetal immune response to hepatitis C virus exposure in utero.

BACKGROUND: Although the rate of mother-to-child transmission of hepatitis C virus (HCV) is low, the effect of HCV exposure in utero on the fetal immune system is unknown. METHODS: Umbilical cord blood was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women and 8 neonates born to HCV-seronegative women. Cord blood mononuclear cells were analyzed by immunophenotyping and by intracellular cytokine staining after HCV-specific and polyclonal stimulation. Plasma was analyzed for anti-HCV immunoglobulin M (IgM), cytokine/granzyme concentrations, and indoleamine 2,3-dioxygenase (IDO) activity. RESULTS: HCV-exposed neonates had significantly lower levels of regulatory T cells expressing HLA-DR, lower CD4(+) and CD8(+) T cell activation, and lower plasma levels of pro-inflammatory markers than did controls. However, CD4(+) and CD8(+) T cells from HCV-exposed neonates had higher IFN-γ production in response to polyclonal stimulation than did T cells from controls. IDO activity was similar between groups. No HCV-specific T cell responses or anti-HCV IgM were detected in any neonates. CONCLUSIONS: HCV-exposed neonates showed a relative suppression of immune activation and pro-inflammatory markers, which was counterbalanced by an increased production capacity for IFN-γ. These results suggest that HCV encounters the fetal immune system in utero, and alters the balance between suppressive and pro-inflammatory responses.

HIV T-cell immunogen design and delivery.

PURPOSE OF THE REVIEW: Not all T-cell responses against HIV are created equally and responses of certain epitope specificities have been associated with superior control of infection. These insights have spurred the development of a wide range of immunogen sequences, each with particular advantages and limitations. RECENT FINDINGS: We review some of the most advanced designs that have reached or are close to reaching human clinical trials, with a special focus on T-cell immunogen developed for therapeutic use. We also touch upon the importance of how immunogens are delivered and point out the lamentable fact that there is essentially no alignment between different designs and vaccine regimens, which is a major hindrance to accelerated advances in the field. SUMMARY: The design of an immunogen able to induce T-cell responses of adequate specificity and functionality is subject of a wide range of preclinical and clinical studies. Few designs have shown promise to date, but emerging data highlight the critical contribution of specificity to effective antiviral activity in vivo .