Kinetics of Myeloid-Derived Suppressor Cell Frequency and Function during Simian Immunodeficiency Virus Infection, Combination Antiretroviral Therapy, and Treatment Interruption.

During chronic lentiviral infection, poor clinical outcomes correlate both with systemic inflammation and poor proliferative ability of HIV-specific T cells; however, the connection between the two is not clear. Myeloid-derived suppressor cells (MDSC), which expand during states of elevated circulating inflammatory cytokines, may link the systemic inflammation and poor T cell function characteristic of lentiviral infections. Although MDSC are partially characterized in HIV and SIV infection, questions remain regarding their persistence, activity, and clinical significance. We monitored MDSC frequency and function in SIV-infected rhesus macaques. Low MDSC frequency was observed prior to SIV infection. Post-SIV infection, MDSC were elevated in acute infection and persisted during 7 mo of combination antiretroviral drug therapy (cART). After cART interruption, we observed MDSC expansion of surprising magnitude, the majority being granulocytic MDSC. At all stages of infection, granulocytic MDSC suppressed CD4+ and CD8+ T cell proliferation in response to polyclonal or SIV-specific stimulation. In addition, MDSC frequency correlated significantly with circulating inflammatory cytokines. Acute and post-cART levels of viremia were similar, however, the levels of inflammatory cytokines and MDSC were more pronounced post-cART. Expanded MDSC during SIV infection, especially during the post-cART inflammatory cytokine surge, likely limit cellular responses to infection. As many HIV curative strategies require cART interruption to determine efficacy, our work suggests treatment interruption-induced MDSC may especially undermine the effectiveness of such strategies. MDSC depletion may enhance T cell responses to lentiviral infection and the effectiveness of curative approaches.

Effects of therapeutic vaccination on the control of SIV in rhesus macaques with variable responsiveness to antiretroviral drugs.

A therapeutic vaccine that induces lasting control of HIV infection could eliminate the need for lifelong adherence to antiretroviral therapy. This study investigated a therapeutic DNA vaccine delivered with a single adjuvant or a novel combination of adjuvants to augment T cell immunity in the blood and gut-associated lymphoid tissue in SIV-infected rhesus macaques. Animals that received DNA vaccines expressing SIV proteins, combined with plasmids expressing adjuvants designed to increase peripheral and mucosal T cell responses, including the catalytic subunit of the E. coli heat-labile enterotoxin, IL-12, IL-33, retinaldehyde dehydrogenase 2, soluble PD-1 and soluble CD80, were compared to mock-vaccinated controls. Following treatment interruption, macaques exhibited variable levels of viral rebound, with four animals from the vaccinated groups and one animal from the control group controlling virus at median levels of 103 RNA copies/ml or lower (controllers) and nine animals, among all groups, exhibiting immediate viral rebound and median viral loads greater than 103 RNA copies/ml (non-controllers). Although there was no significant difference between the vaccinated and control groups in protection from viral rebound, the variable virological outcomes during treatment interruption enabled an examination of immune correlates of viral replication in controllers versus non-controllers regardless of vaccination status. Lower viral burden in controllers correlated with increased polyfunctional SIV-specific CD8+ T cells in mesenteric lymph nodes and blood prior to and during treatment interruption. Notably, higher frequencies of colonic CD4+ T cells and lower Th17/Treg ratios prior to infection in controllers correlated with improved responses to ART and control of viral rebound. These results indicate that mucosal immune responses, present prior to infection, can influence efficacy of antiretroviral therapy and the outcome of immunotherapeutic vaccination, suggesting that therapies capable of modulating host mucosal responses may be needed to achieve HIV cure.

Mucosal T Helper 17 and T Regulatory Cell Homeostasis Correlate with Acute Simian Immunodeficiency Virus Viremia and Responsiveness to Antiretroviral Therapy in Macaques.

Depletion of gut T helper 17 (Th17) cells during HIV infection leads to decreased mucosal integrity and increased disease progression. Conversely, T regulatory (Treg) cells may inhibit antiviral responses or immune activation. In HIV elite controllers, a balanced Th17/Treg ratio is maintained in the blood, suggesting a role for these responses in controlling inflammation and viral replication. HIV-infected individuals exhibit a range in responsiveness to combination antiretroviral therapy (cART). Given the link between the Th17/Treg ratio and HIV disease, we reasoned these responses may play a role in cART responsiveness. In this study, we investigated the relationship between the mucosal Th17/Treg ratio to acute simian immunodeficiency virus (SIV) viremia and the response to cART. Nineteen rhesus macaques were infected with highly pathogenic SIVΔB670 virus and cART was initiated 6 weeks postinfection. Mucosal CD4 T cell subsets were assessed by intracellular cytokine staining in the colon and mesenteric lymph nodes. Higher baseline Th17/Treg ratios corresponded with increased acute SIV viremia. Th17/Treg ratios decreased during acute SIV infection and were not restored during cART, and this corresponded to increased gut immune activation (Ki67+), markers of microbial translocation (sCD14), and T cell exhaustion (TIGIT+). Animals that maintained a more balanced mucosal Th17/Treg ratio at the time of cART initiation exhibited a better virological response to cART and maintained higher peripheral CD4 counts. These results suggest mucosal Th17 and Treg homeostasis influences acute viremia and the response to cART, a result that suggests therapeutic interventions that improve the Th17/Treg ratio before or during cART may improve treatment of HIV.

Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection.

HIV-specific T-cell responses play a key role in controlling HIV infection, and therapeutic vaccines for HIV that aim to improve viral control will likely need to improve on the T-cell responses induced by infection. However, in the setting of chronic infection, an effective therapeutic vaccine must overcome the enormous viral genetic diversity and the presence of pre-existing T-cell responses that are biased toward immunodominant T-cell epitopes that can readily mutate to evade host immunity and thus potentially provide inferior protection. To address these issues, we investigated a novel, epidermally administered DNA vaccine expressing SIV capsid (p27Gag) homologues of highly conserved elements (CE) of the HIV proteome in macaques experiencing chronic but controlled SHIV infection. We assessed the ability to boost or induce de novo T-cell responses against the conserved but immunologically subdominant CE epitopes. Two groups of animals were immunized with either the CE DNA vaccine or a full-length SIV p57gag DNA vaccine. Prior to vaccination, CE responses were similar in both groups. The full-length p57gag DNA vaccine, which contains the CE, increased overall Gag-specific responses but did not increase CE responses in any animals (0/4). In contrast, the CE DNA vaccine increased CE responses in all (4/4) vaccinated macaques. In SIV infected but unvaccinated macaques, those that developed stronger CE-specific responses during acute infection exhibited lower viral loads. We conclude that CE DNA vaccination can re-direct the immunodominance hierarchy towards CE in the setting of attenuated chronic infection and that induction of these responses by therapeutic vaccination may improve immune control of HIV.

Early cellular innate immune responses drive Zika viral persistence and tissue tropism in pigtail macaques.

The immunological and virological events that contribute to the establishment of Zika virus (ZIKV) infection in humans are unclear. Here, we show that robust cellular innate immune responses arising early in the blood and tissues in response to ZIKV infection are significantly stronger in males and correlate with increased viral persistence. In particular, early peripheral blood recruitment of plasmacytoid dendritic cells and higher production of monocyte chemoattractant protein (MCP-1) correspond with greater viral persistence and tissue dissemination. We also identify non-classical monocytes as primary in vivo targets of ZIKV infection in the blood and peripheral lymph node. These results demonstrate the potential differences in ZIKV pathogenesis between males and females and a key role for early cellular innate immune responses in the blood in viral dissemination and ZIKV pathogenesis.

Expression of LHX3 and SOX2 during mouse inner ear development.

A cascade of transcription factors is believed to regulate the coordinate differentiation of primordial inner ear cells into the subtypes of hair cells and supporting cells. While candidate genes involved in this process have been identified, the temporal and spatial patterns of expression of many of these have not been carefully described during the extended period of inner ear development and functional maturation. We systematically examined the expression of two such transcription factors, LHX3 and SOX2, from the time of hair cell terminal mitoses into adulthood. We show that LHX3 is expressed specifically in auditory and vestibular hair cells soon after terminal mitoses and persists into the adult in vestibular hair cells. While SOX2 expression is widespread in the inner ear sensory epithelia prior to hair cell differentiation, it has a unique pattern of expression in the mature auditory and vestibular organs.

Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates.

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains.

Prospectively-collected, tooth-specific dosimetry correlated with adverse dental outcomes.

OBJECTIVES: To correlate radiation dose to specific tooth-bearing portions of bone with adverse dental outcomes. STUDY DESIGN: Eighty-nine patients treated with intensity-modulated radiation therapy with or without chemotherapy had radiation dose to specific tooth-bearing portions of the mandible and the maxilla. Data were collected prospectively during treatment planning, which resulted in 2490 data points. These patients underwent a comprehensive dental intake evaluation that included measurement of pocket depths and were then followed up with serial dental evaluations for a median of 2.5 years (range 0.2-6.9 years). RESULTS: At the patient level, the 3-year risks of osteoradionecrosis (ORN) and periodontal disease were 2.5% and 36.6%, respectively. For any individual tooth, the risks of ORN and periodontal disease were 0.1% and 5.1%, respectively, at 3 years. Radiation dose to individual tooth-bearing portions of bone was correlated with ORN development (P = .0165). Periodontal disease also demonstrated a significant, but more gradual, dose response (P = .0395). CONCLUSIONS: Adverse dental outcomes directly correlate with increased tooth-specific doses.

Use of glnQ as a counterselectable marker for creation of allelic exchange mutations in group B streptococci.

Efficient allelic exchange mutagenesis in group B streptococci (GBS) has been hampered by the lack of a counterselectable marker system. Growth inhibition of GBS by the glutamine analog gamma-glutamyl hydrazide requires glnQ. We have used this phenomenon to create a counterselectable marker system for efficient selection of allelic exchange mutants in GBS.