Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

Abstract

Virulent strains of Rhodococcus equi have a large plasmid of 80-90kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2(-/-) mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4(+) T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4(+) T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.

Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Rhodococcus equi Infection through Induced Humoral Immunity

“…However, the TNF-α content decreased when the immunized mice were challenged with R. equi , reaching to levels comparable to those in the mice from the negative control group ( Figure 2F ). Notably, the control group vaccinated with the empty vector ( Salmonella-vapA -) augmented the TNF-α content in the spleen significantly, and this result was consistent with that of the previous studies (Oliveira et al, 2010; Cardoso et al, 2013). …”

“…It was previously demonstrated that oral immunization of mice with Salmonella - vapA + confers resistance toward R. equi infection (Oliveira et al, 2007, 2010; Cardoso et al, 2013). Because VapG antigen, similar to VapA, is encoded by a gene of the R. equi virulence plasmid and is highly expressed in the lung tissue of R. equi -infected foals (Coulson et al, 2010), in this study, we evaluated the effect of Salmonella - vapG + vaccination in mice.…”

“…In the present study, we assessed VapG as a Salmonella -based live-vector vaccine, an approach that was previously efficient in delivering VapA (Oliveira et al, 2007, 2010; Cardoso et al, 2013). Vaccination with Salmonella - vapG + significantly reduced the bacterial burden exhibited by R. equi -challenged mice.…”

Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages

“…In addition, CR3 -/- mice infected with virulent M. tuberculosis did not differ in tissue infection levels or time of death compared to WT ( 55 ). TLR2 -/- mice have shown similar susceptibility to virulent R. equi infection and ability to respond to immunization ( 20 ). Together, these data suggest that TLR2 and CR3 are important in controlling the replication of avirulent R. equi , but the pathways downstream of these receptors fail to control virulent R. equi replication.…”

“…TLR2 recognizes pathogen-associated molecular patterns present in R. equi , such as lipoteichoic acid, lipoarabinomannan, among others ( 17 – 19 ), and is activated by infection with R. equi or rVapA alone, inducing pro-inflammatory cytokine expression in macrophages ( 15 ). There is conflicting evidence about the importance of TLR2 for killing R. equi in mice: TLR2 -/- mice failed to clear virulent R. equi infection ( 15 ), whereas TLR2 -/- mice that received a VapA vaccine were protected against R. equi infection ( 20 ). This vaccine, however, contained an attenuated Salmonella expressing VapA, and other factors such as non-specific stimulation of the innate immune system ( 21 ) could play a role in protection.…”