Rabies is a zoonotic disease that continues to be an important public health problem causing an estimated 55,000 human deaths each year globally, the majority of which occur in Africa and Asia (1
). In Asia, parts of America, and large parts of Africa, canines remain the principal host of rabies virus (RABV), and more than 95% of all human rabies cases are caused by exposure to infected dogs (2
). The most effective means to protect humans and livestock against rabies is by prophylactic immunization with an RABV vaccine. Although inactivated tissue culture RABV vaccines are safe, they induce protective immunity only when administered in multiple doses and generally require repeat booster doses to provide long-lasting protection. Repeated prophylactic immunization is cost-prohibitive and operationally unrealistic for both people and animals in developing countries. A single-dose RABV vaccine capable of inducing robust, enduring immunity would be highly advantageous in controlling rabies in dogs, livestock, and humans worldwide.
A hallmark of wild-type RABV is its neuroinvasiveness—a unique ability to invade the central nervous system (CNS) from peripheral sites of inoculation (4
). Important aspects of this property include the capacity to preserve the integrity of the neuronal network and the ability to evade host immunity (6
). One of the important mechanisms that contribute to both of these features of rabies pathogenesis is the control of viral replication, which is seen with pathogenic but not attenuated RABV strains (8
). Low replication levels contribute to RABV pathogenesis by conserving the structure of the neurons that are used by these viruses to spread to the CNS and by minimizing viral antigen exposure to the host immune system (10
). This is particularly important for RABV glycoprotein (G protein), which is the major target of RABV-specific immunity (11
). In contrast to wild-type RABVs, attenuated RABV strains replicate rapidly, express large amounts of G protein, and induce strong innate and adaptive immune responses that can clear an RABV infection. These properties form the basis for the utility of attenuated RABV strains in pre- and postexposure prophylaxis against wild-type RABV infections.
Studies of antibody escape mutants revealed that the main contributor to the pathogenicity of RABV is its G protein, in that substitution of the Arg at position 333 with Glu renders an RABV considerably less pathogenic (13
). Reverse genetics technology has been used to construct SADB19-based recombinant RABVs with this substitution together with an Asn194-to-Ser194 mutation that stabilizes the attenuated phenotype (8
). Although recombinant RABVs containing this doubly modified G, termed GAS, are strongly attenuated, we have found that engineering the virus to express additional GAS genes not only augments its nonpathogenic phenotype but also substantially increases its immunogenicity (11
). While single and double GAS variants are largely apathogenic for normal adult mice, they are cytopathic in vitro
and have residual pathogenicity for immunocompromised animals (11
). Containing three GAS genes, the TriGAS RABV variant is unique in being nonpathogenic for mice that either are developmentally immunocompromised or have inherited deficits in immune functions (17
). Evidence suggests that the higher level of attenuation of TriGAS is related to increased G expression rather than an increase in genome size (17
). This is accompanied by an enhanced capacity to induce protective RABV immunity, which is best illustrated by the fact that TriGAS inoculation provides prophylactic protection to mice several hours to days after infection with a lethal dose of wild-type RABV (17
The unique capacity of superinfection with TriGAS to prevent lethal rabies in mice infected with wild-type RABV suggests that this attenuated virus induces protective elements of immunity considerably more rapidly than other variants. RABV-neutralizing antibodies (VNAs) are the major immune effectors against RABV, and rabies G is the target for nearly all VNAs (21
). As expected from their role in promoting antibody production, the activity of CD4 T cells is critical to the development of protective immunity against rabies (22
). The presentation of RABV antigen to these cells is therefore one of several rate-limiting steps in the induction of RABV immunity. During RABV infection, the rate of RABV RNA synthesis and G expression correlate with changes in the expression levels of a variety of host genes (8
). This indicates that the products of RABV infection, including viral RNA and G protein, likely include pathogen-associated molecular patterns (PAMPs), which are recognized by pathogen recognition receptors (PRRs) such as RIG-I-like helicases and toll-like receptors (TLRs) (26
). For example, mice lacking TLR7 exhibit a delay in the production of RABV-specific antibodies and increased mortality when infected with RABV (20
). The induction of PRRs generally depends on virus replication (26
), and this is evidently also the case for RABV, for which infection induces a TH1-biased response, as opposed to the TH2-biased response elicited by the administration of killed vaccine (19
). Thus, there are several features that suggest that the induction of RABV immunity may be superior with live vaccines, such as TriGAS, than inactivated vaccines.
Although live-attenuated rabies vaccines have been extensively used for oral immunization of wildlife, their use in domestic animals and, particularly, humans must be approached cautiously. The possibility that an attenuated RABV might spread to the CNS and replicate in CNS tissues at low levels that evade immune detection, thereby causing progressive neuronal damage, must be considered. Therefore, to provide further insight into the safety and efficacy of TriGAS, we carefully determined both the location and time course of TriGAS infection after intramuscular (i.m.) administration. We demonstrate that TriGAS infects particular immune cell types, without detectable virus propagation, in the lymph nodes draining the site of inoculation. Infection of these cells orchestrates potent, enduring immune protection against rabies.