West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4 + Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA + ) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.

Intermittent low-level HCV viremia can occur as much as 2 months before the periods of exponential increase in viral load and the high-titer plateau-phase viremia that usually precede seroconversion. Animal inoculation studies are in progress to evaluate if transfusion of low-level viremic plasma can transmit HCV infection.

Objective HIV+ elite controllers are a unique group of rare individuals who maintain undetectable viral loads in the absence of antiretroviral therapy. We studied immune responses in these subjects to inform vaccine development, with the goal of identifying the immune correlates of protection from HIV. Methods We compared markers of cellular activation, HIV-specific immune responses, and regulatory T (Treg) cell frequencies in 4 groups of subjects: HIV-negative healthy controls, elite controllers (HIV RNA level <75 copies/ml), individuals on highly active antiretroviral therapy (HAART), and subjects with HIV RNA level >10,000 copies/ml (non-controllers). Results Elite controllers possessed significantly lower levels of activated HIV-specific CD8+ T cells and of recently divided HIV-specific CD4+ T cells than non-controllers, while these differences were not seen in the respective CMV-specific T cell populations. Elite controllers also mounted a stronger and broader cytokine and chemokine response following HIV-specific stimulation than individuals on HAART and non-controllers. Finally, we found that HAART suppressed subjects had elevated Treg cell frequencies, while elite controllers and non-controllers maintained normal percentages of Treg cells. Conclusion Elite controllers maintain high levels of HIV-specific immune responses with low levels of HIV-specific T cell activation, and do not have elevated Treg cell levels. Based on these data an ideal HIV vaccine would induce strong HIV-specific immune responses while minimizing HIV-specific T cell activation.

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined.Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4 + T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8 + T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8 + T cell responses to peptides and peptide pools were well preserved. Loss of both CD4 + and CD8 + T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4 + T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein.Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4 + T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4 + and CD8 + T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4 + T cell responses and mild skewing of T cell phenotypic marker expression.

Differences in sensitivities of licensed NAT assays for HIV and HCV are very small and clinically insignificant, particularly when compared to differences of MP versus ID NAT screening.