Obligatory requirement for antibody in recovery from a primary poxvirus infection

Abstract

To understand the correlates of protective immunity against primary variola virus infection in humans, we have used the well-characterized mousepox model. This is an excellent surrogate small-animal model for smallpox in which the disease is caused by infection with the closely related orthopoxvirus, ectromelia virus. Similarities between the two infections include virus replication and transmission, aspects of pathology, and development of pock lesions. Previous studies using ectromelia virus have established critical roles for cytokines and effector functions of CD8 T cells in the control of acute stages of poxvirus infection. Here, we have used mice deficient in B cells to demonstrate that B-cell function is also obligatory for complete virus clearance and recovery of the host. In the absence of B cells, virus persists and the host succumbs to infection, despite the generation of CD8 T-cell responses. Intriguingly, transfer of naive B cells or ectromelia virus-immune serum to B-cell-deficient mice with established infection allowed these animals to clear virus and fully recover. In contrast, transfer of ectromelia virus-immune CD8 T cells was ineffective. Our data show that mice deficient in CD8 T-cell function die early in infection, whereas those deficient in B cells or antibody production die much later, indicating that B-cell function becomes critical after the effector phase of the CD8 T-cell response to infection subsides. Strikingly, our results show that antibody prevents virus from seeding the skin and forming pock lesions, which are important for virus transmission between hosts.

FIG. 1.

Survival and virus load in organs of mice infected with ECTV. (A) Groups of 10 B6.WT, B6.IFN-γ^−/−, B6.Prf^−/− or B6.Aa^−/− mice and a group of 13 B6.μMT mice were infected with 10³ PFU of ECTV and monitored for clinical signs of disease and survival. (B) In a separate experiment, groups of six B6.WT, B6.IFN-γ ^−/−, B6.Prf^−/−, or B6.Aa^−/− mice and a group of eight B6.μMT mice were infected as described for panel A and sacrificed at day 7 p.i., and organs were collected. Viral titers in spleen and liver were determined and are presented as means ± standard deviations.

FIG. 2.

ECTV infection causes pock lesions and conjunctivitis in the absence of B cells and antibody. ECTV-infected B6.WT mice do not develop rashes, lesions, or conjunctivitis. From day 10 to 14 p.i., ECTV-infected B6.μMT mice develop conjunctivitis (red arrow), lesions (white arrow), and inflammation (blue arrow) on the pinnae and lesions on the tail (black arrows). A representative B6.WT mouse and a B6.μMT mouse at 14 days p.i. are shown.

FIG. 3.

Splenic ECTV-specific CTL activity. Groups of five B6.WT, B6.IFN-γ^−/−, B6.Prf^−/−, B6.Aa^−/− or B6.μMT mice were infected with 10³ PFU of ECTV. At day 7 p.i., mice were sacrificed and splenic ECTV-specific CTL activity was measured ex vivo using ECTV-infected and uninfected MC57G target cells.

FIG. 4.

Persistence of virus and anti-ECTV CTL activity in B6.μMT and B6.Aa^−/− mice. B6.WT, B6.μMT, and B6.Aa^−/− mice were infected with 10³ PFU of ECTV, three animals from each group were sacrificed at 24 days p.i., and various tissues were removed. (A) Viral titers in spleen, liver, lung, pinnae, and tail tissues were determined. The broken line indicates the limit of detection of the assay, which for these experiments was 100 PFU. No virus was detected in any tissues from B6.WT mice. (B) Splenic anti-ECTV-specific CTL activity was measured ex vivo as for Fig. 3.

FIG. 5.

Kinetics of ECTV-neutralizing antibody response and viremia. (A) Groups of B6.WT mice were infected with 10³ PFU of ECTV, and at various times (days 7 [designated D7], 10 [D10], 14 [D14], 17 [D17], and 31 [D31] p.i), five mice from each group were bled and sera collected. Sera were also collected from control, uninfected (D0) mice. The kinetics of the neutralizing antibody response in sera of mice for each time point was measured by the plaque reduction neutralization test. The neutralizing titer was taken to be the reciprocal of the serum dilution at which 50% of the virus was neutralized. There was no virus-neutralizing activity in sera of uninfected (D0) mice. (B) In a separate experiment, B6.WT or B6.μMT mice were infected with 10³ PFU of ECTV, and at various times p.i., five mice from each group were bled and viremia determined by virus plaque assay. The broken line indicates the limit of detection of the assay.

FIG. 6.

Transfer of naive B cells or ECTV-immune serum to B6.μMT mice. B6.μMT mice were infected with 10³ PFU of ECTV, and after the infection was established, groups of 10 mice were left untreated, given ECTV-immune serum from B6.WT mice at day 10 p.i., or given 1 × 10⁷ of the following purified cell subsets: naive B6.WT B cells, naive B6.Aa^−/− B cells, or ECTV-immune B6.WT CD8 T cells. Mice were monitored for clinical signs of disease, and all surviving mice were sacrificed at day 35 p.i.