The Orchestrated Functions of Innate Leukocytes and T Cell Subsets Contribute to Humoral Immunity, Virus Control, and Recovery from Secondary Poxvirus Challenge

Abstract

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells.However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infection. S mallpox, caused by variola virus, was considered among the deadliest scourges of humankind. It was eradicated more than 30 years ago through one of the most successful immunization campaigns, which employed a vaccine containing the closely related vaccinia virus (VACV). Although the VACV strain used in the smallpox vaccine is not considered safe by current standards, it was potent in inducing long-lived memory and offered a high degree of protection. Much of our current understanding of protection following vaccination and recall responses to secondary challenge has been inferred from animal studies of closely related poxvirus infections, including mousepox (a disease caused by ectromelia virus [ECTV] in mice), VACV, and monkeypox.We have shown previously that neutralizing antibody, but not the function of CD4 or CD8 T cell subsets, is required to control virus replication during the acute phase of a secondary ECTV challenge (1). In a separate study on monkeypox, depletion of CD4 or CD8 T cells also had no significant effect on virus clearance or on neutralizing antibody production during the acute phase of a secondary challenge in macaques vaccinated with VACV vaccine 6 months previously (2). In both studies, neutralizing antibody produced in the absence of CD4 T cell help (attributed to extrafollicular plasma cells) was sufficient for virus control in immune animals. A number of other studies have found that in vaccinated individuals, humoral immunity to smallpox is stable and lasts longer than memory CD4 and CD8...

Vaccine-Induced Protection against Orthopoxvirus Infection Is Mediated through the Combined Functions of CD4 T Cell-Dependent Antibody and CD8 T Cell Responses

“…Recent evidence from our laboratory indicates that innate immunity also plays a key role during the first 8 days p.c. NK cells, granulocytes, and plasmacytoid dendritic cells are important for virus control, and their essential roles become apparent in the absence of CD4 and CD8 T cell subsets (41). In the current study, through prolonged CD4 T cell depletion, we have established that CD4 T cell help is essential for a GC response in order to sustain production of high titers of neutralizing antibody as the infection progresses.…”

“…Virus titers, expressed as log 10 PFU per gram tissue, were determined on BS-C-1 monolayers using the conventional viral plaque assay, as described previously (13,38). Low viral titers undetectable by viral plaque assay and the virus load in blood were measured by quantitative real-time PCR (qRT-PCR), as described previously (41). The viral genome copy number was correlated with the copy number of the late gene ECTV-Mos-156, which encodes the viral hemagglutinin (42).…”

“…PRNT. The plaque reduction neutralization test (PRNT), used to determine the virus-neutralizing activity of the antibody present in serum samples, was described previously (25,27,41). Sera were heat inactivated at 56°C for 30 min prior to use.…”

“…ECTV-specific IgG and subtypes were quantified by enzyme-linked immunosorbent assay (ELISA), as described previously (25,27,41). The enzyme-linked immunospot assay (ELISPOT) for quantifying ECTV-specific ASC has been described in detail previously (41).…”

“…At 5 to 16 weeks postvaccination with ECTV-TK ⌬ , the neutralizing antibody titers are very low or below the level of detection. However, by 8 days p.c., high levels of neutralizing antibody are detectable (41).…”