Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement

Abstract

Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV).Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.Vaccines are one of the most cost-effective medical treatments in modern civilization (80). A smallpox vaccine was the first human vaccine, and the modern smallpox vaccine, live vaccinia virus (VACV), is the most successful human vaccine, bringing about the worldwide eradication of smallpox disease due to a heroic World Health Organization campaign in the 1960s and 1970s (27). Elucidating the immunobiology underlying the protection provided by the smallpox vaccine will continue to reveal vaccinology principles that can be applied to future vaccine development against other infectious scourges (95). Neutralizing antibodies are of primary importance in the protection from smallpox provided by the smallpox vaccine in animal models (6,25,33,64) and humans (4). Vaccinia immune globulin (VIG) has been shown to be an effective treatment against smallpox (45) as it was able to reduce the number of smallpox cases by ϳ80% among exposed individuals in four case-controlled studies (42,45,50,51,66).Neutralizing antibodies confer protection mainly through the recognition of structures on the surface of virus particles, and therefore antiviral antibodies directed against the surface of virions are of primary inter...

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

“…The modest decrease in titer with the heat-inactivated serum was also previously observed with vaccinia virus (6). The relatively equivalent results after exposing EEV to either active or heat-inactivated serum establishes the resistance of the EEV form to complement-mediated neutralization in a second virus.…”

The Vaccinia Virus Complement Control Protein Modulates Adaptive Immune Responses during Infection

“…Benhnia et al showed that monoclonal Abs (MAbs) specific for the EV protein B5 that are able to initiate complement-mediated lysis of infected cells and complement-dependent neutralization of EV particles in vitro provide better protection against VACV challenge following passive immunization (2,3). Furthermore, the enhanced protection provided by these MAbs in vivo was reduced when mice that had been passively immunized were depleted of complement prior to challenge, demonstrating that enhanced protection depended on the ability of MAbs to activate complement (3). Our data demonstrate that VCP limits the early nAb antibody response, but it is also likely that VCP reduces complement-mediated neutralization by nAbs that are produced.…”

Poorly Neutralizing Cross-Reactive Antibodies against the Fusion Loop of West Nile Virus Envelope Protein ProtectIn Vivovia Fcγ Receptor and Complement-Dependent Effector Mechanisms

“…For preparation of splenocytes as single-cell suspensions, spleens were manually homogenized on 40-m cell strainers into RPMI medium (Sigma). For preparation of peripheral blood leukocytes (PBL), blood was collected into EDTA-coated tubes, and erythrocytes were lysed by diluting whole blood in ACK buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA) for 5 min. Splenocytes or PBL were washed thrice in PBS with 2% FBS prior to staining with cell-type-specific conjugated MAbs for 30 min on ice.…”