Although naturally occurring smallpox was eliminated through the efforts of the World Health Organization Global Eradication Program, it remains possible that smallpox could be intentionally released. Here we examine the magnitude and duration of antiviral immunity induced by one or more smallpox vaccinations. We found that more than 90% of volunteers vaccinated 25-75 years ago still maintain substantial humoral or cellular immunity (or both) against vaccinia, the virus used to vaccinate against smallpox. Antiviral antibody responses remained stable between 1-75 years after vaccination, whereas antiviral T-cell responses declined slowly, with a half-life of 8-15 years. If these levels of immunity are considered to be at least partially protective, then the morbidity and mortality associated with an intentional smallpox outbreak would be substantially reduced because of pre-existing immunity in a large number of previously vaccinated individuals.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19−CD38hiCD138+ subset was morphologically distinct, differentially expressed PC-associated genes and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for over 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19−CD38hiCD138+ PCs in the BM. Finally, we found that CD19−CD38hiCD138+ PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and likely represents the B cell response’s “historical record” of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Approximately 50% of the US population received smallpox vaccinations before routine immunization ceased in 1972 for civilians and in 1990 for military personnel. Several studies have shown long-term immunity after smallpox vaccination, but skepticism remains as to whether this will translate into full protection against the onset of orthopoxvirus-induced disease. The US monkeypox outbreak of 2003 provided the opportunity to examine this issue. Using independent and internally validated diagnostic approaches with >or=95% sensitivity and >or=90% specificity for detecting clinical monkeypox infection, we identified three previously unreported cases of monkeypox in preimmune individuals at 13, 29 and 48 years after smallpox vaccination. These individuals were unaware that they had been infected because they were spared any recognizable disease symptoms. Together, this shows that the US monkeypox outbreak was larger than previously realized and, more importantly, shows that cross-protective antiviral immunity against West African monkeypox can potentially be maintained for decades after smallpox vaccination.

Virus-specific CD8 + T cells develop the ability to function in an "innate" capacity by responding to a remarkable array of cytokines in a TCR-independent manner. Although several cytokines such as IL-12 and IL-18 have been identified as key regulators of CD8 + T-cell activation, the role of other cytokines and the ways in which they interact with each other remain unclear. Here, we have used an unbiased, systematic approach to examine the effects of 1,849 cytokine combinations on virus-specific CD8 + T-cell activation. This study identifies several unexpected cytokine combinations that synergize to induce antigen-independent IFNγ production and CD69 up-regulation by CD8 + T cells in addition to cytokines that exhibit differential regulatory functions, with the ability to either enhance or inhibit T-cell IFNγ production, depending on which cytokine partner is present. These findings underscore the complexity of cytokine interactions while also providing insight into the multifaceted regulatory network controlling virusspecific CD8 + T-cell functions.lymphocytic choriomeningitis virus | mouse | interleukin | lymphocyte

Pre-existing serum antibodies play an important role in vaccine-mediated protection against infection but the underlying mechanisms of immune memory are unclear. Clinical studies indicate that antigen-specific antibody responses can be maintained for many years, leading to theories that reactivation/differentiation of memory B cells into plasma cells is required to sustain long-term antibody production. Here, we present a decade-long study in which we demonstrate site-specific survival of bone marrow-derived plasma cells and durable antibody responses to multiple virus and vaccine antigens in rhesus macaques for years after sustained memory B cell depletion. Moreover, BrdU+ cells with plasma cell morphology can be detected for 10 years after vaccination/BrdU administration, indicating that plasma cells may persist for a prolonged period of time in the absence of cell division. On the basis of these results, long-lived plasma cells represent a key cell population responsible for long-term antibody production and serological memory.

Monkeypox virus (MPV) is a virulent human pathogen that has gained increased attention because of its potential use as a bioterrorism agent and inadvertent introduction into North America in 2003. The US outbreak also provided an important opportunity to study MPV-specific T cell immunity. Although MPV-specific CD4 ؉ and CD8 ؉ T cells could recognize vaccinia virus (VV)-infected monocytes and produce inflammatory cytokines such as IFN␥ and TNF␣, they were largely incapable of responding to autologous MPV-infected cells. Further analysis revealed that, unlike cowpox virus (CPV), MPV did not interfere with MHC expression or intracellular transport of MHC molecules. Instead, MPV-infected cells were capable of preventing T cell receptor (TcR)-mediated T cell activation in trans. The ability to trigger a state of nonresponsiveness represents a unique MHC-independent mechanism for blocking antiviral T cell activation and inflammatory cytokine production and is likely an important attribute involved with viral dissemination in the infected host. Although MPV does not spread efficiently by human-to-human contact (12)(13)(14), it serves as an important model for smallpox (15-18) and shares several key features of pathogenesis. For instance, unlike vaccinia (VV) (19), both VAR and MPV disseminate through their infected hosts mainly by a cell-associated viremia (15,(20)(21)(22)(23). Moreover, evasion of host immune responses is well documented; VAR infection of previously vaccinated humans and MPV infection of non-human primates can result in infectious virus persisting for prolonged periods of time as an asymptomatic infection in apparently healthy individuals (24-32).The mechanisms underlying these forms of immune evasion are not well understood. Many viruses employ a battery of immune evasion strategies (33-38) and poxviruses in particular are equipped to evade antiviral cytokines, chemokines, and/or antigen presentation (35, 39). We have shown that cowpox virus (CPV) interferes with intracellular transport of MHC class I, a process that correlated with evasion of antiviral CD8 ϩ T cell responses by CPV (40). It was recently demonstrated that CPV open reading frame 203 retains MHC class I in the ER (41), and, because MPV encodes a close homologue of CPV203, we expected to find a similar mechanism of immune evasion by MPV. In contrast, we observed that MPV did not down-regulate MHC class I, but instead used a mechanism of evasion that inhibited CD4 ϩ and CD8 ϩ T cell activation after cognate interactions with MPV-infected cells. This mechanism of abrogating local T cell responses may avoid systemic immune suppression, while at the same time protecting the viral reservoir from immune surveillance. Identification of the factor or factors involved with MPV-induced T cell inhibition could prove useful for developing new biologics aimed at preventing or alleviating T cell-mediated diseases (42, 43). Results Antiviral CD4 ؉ and CD8 ؉ T Cells Recognize VV-Infected Monocytes butNot MPV-Infected Monocytes. Several human HLA-bindi...

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-γ production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8+ T cells directly ex vivo. Following acute viral infection, 70–80% of virus-specific T cells will produce IFN-γ after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-γ production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.

SUMMARY Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe poxvirus infections, but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal Abs from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.