Although the incidence of HPS cases in North and South America remains low in comparison to that of HFRS cases in Europe and Asia, the significantly higher case fatality rate and the lack of any licensed vaccines or effective treatments for the disease suggest an urgent need for the development of such measures (29
). The hantavirus glycoproteins are typically thought to be the principal targets of neutralizing antibodies (1
), and although a few potential vaccine candidates have been evaluated with various degrees of success, largely in infection rather than disease models (2
), the mechanism of protection against hantavirus infections remain unidentified. Here we constructed an attenuated, replication-competent recombinant VSV expressing the ANDV GPC in order to investigate its efficacy as a hantavirus vaccine in the Syrian hamster HPS disease model and to gain further insight into the mechanism of protection against lethal ANDV infection.
The VSV vector expressing the ANDV GPC () represents a unique attenuated replication-competent VSV construct that expresses a glycoprotein from a bunyavirus, although replication-incompetent VSV particles have previously been pseudotyped with glycoproteins from “Old World” (35
) and “New World” (54
) hantaviruses. Bunyaviruses are generally believed to mature and bud from the Golgi apparatus, including the incorporation of the glycoproteins into virus particles (63
). This is in contrast to the VSV egress pathway, where glycoprotein incorporation and particle budding occur at the plasma membrane (40
). Thus, the ability to rescue the VSV containing the ANDV glycoproteins seems to indicate that at least a portion of the ANDV glycoproteins are transported to the plasma membrane, a mechanism similar to what has been reported before for the glycoproteins of different “Old World” (Hantaan, Seoul) and “New World” (Black Creek Canal) hantaviruses (49
). The incorporation of a foreign transmembrane glycoprotein into VSV particles is likely further reduced by a less optimal interaction of these foreign proteins with VSV structural proteins, such as the M protein, as has been shown for the incorporation of other type I transmembrane glycoproteins into VSV particles in the absence of VSV G (15
The recombinant VSVΔG/ANDVGPC was able to fully protect hamsters with a single dose of vaccine (A and B). Concerns have been raised about the use of replication-competent VSV vaccines, particularly in immunosuppressed patients, such as those treated with immunosuppressive drugs or infected with human immunodeficiency virus (HIV). However, rhesus macaques infected with simian-human immunodeficiency virus (SHIV) showed no adverse effect following immunization with a replication-competent VSV expressing the Ebola virus glycoprotein (VSVΔG/ZEBOVGP), and the vaccine was still able to protect 66% of the highly immunocompromised animals against lethal Ebola virus challenge (16
). In addition, the vaccine dose of replication-competent vaccines is expected to be much lower (approximately 3 log units) and thus less toxic than that of the replication-incompetent Ad5 platform, based on recent studies that used both platforms as Ebola virus vaccines (18
). Another advantage of the VSV vaccines is the negligible level of preexisting neutralizing antibodies against VSV G in the world population (40
). Furthermore, G-neutralizing antibodies are unlikely to affect VSV vaccine efficacy if these vaccines are based on VSV G deletion vectors, as in our study here. Finally, there seems to be a lack of serious pathogenicity in humans associated with VSV infections (4
), making VSV altogether a very suitable candidate for a vaccine platform.
A previous vaccination strategy using DNA vaccination of hamsters with ANDV M-segment cDNA was unable to produce an immune response that afforded protection against lethal ANDV challenge (10
). However, the injection of the same DNA vaccine into nonhuman primates or rabbits was able to generate a humoral immune response that, when passively transferred into hamsters, fully protected them from lethal challenge (24
). This is partially consistent with our data, which suggest that the production of a strong neutralizing antibody response may be sufficient to protect from lethal disease when individuals are immunized at 28 or 14 days before challenge. We found that high levels of neutralizing antibodies were produced at 28 days after immunization, with low to moderate levels produced at 14 days. However, no neutralizing antibodies were detected prior to challenge at either 7 or 3 days after immunization (B). Humoral immune responses have also been viewed as important for protection in other hantavirus studies using small-animal infection models (25
) as well as mild clinical cases of HPS in humans (3
). In contrast, the recent success with the Ad5-based vaccines expressing GN
alone or in combination with GC
seems to be attributed mainly to a strong cellular immune response (mainly CD8+
cytotoxic lymphocytes), as observed in BALB/c mice (60
). Unfortunately, no Ad5 expressing the full-length ANDV GPC was investigated, preventing us from making a direct comparison of the immune response between the Ad5 and VSV platforms. Further, immune reagents with which to examine the hamster adaptive cellular responses have been sorely lacking, currently limiting the examination of these responses in both the Ad5 (60
) and VSV (this study) vaccine studies.
Interestingly, the humoral immune response does not seem to be important for protection if the vaccine is administered within a week prior to challenge, an application that fully protected the animals from disease and resulted in seemingly limited challenge virus replication (A and A). We propose that a strong nonspecific VSV-mediated innate response is the important mechanism here (40
). It has been shown that hantaviruses are sensitive to innate responses, particularly interferons, prior to the establishment of infection but become insensitive to treatment with interferons once infection is established (68
). Both VSVΔG/ANDVGPC and VSVΔG/ZEBOVGP were shown to stimulate the production of Mx-2, STAT-1, and IFN-γ during this 7-day period (). The strong induction of innate immune responses by VSVΔG/ZEBOVGP may explain why some control hamsters survived when immunized at 3 or 7 days before challenge. However, the fact that all VSVΔG/ANDVGPC-immunized animals survived during the same period suggests that a glycoprotein-specific adaptive immune response may also be occurring.
Previous investigations have shown that recombinant VSV expressing either the Ebola virus or the Marburg virus glycoprotein administered postexposure provided full or partial protection from lethal challenge (12
). Therefore, we also tested the use of VSVΔG/ANDVGPC as a postexposure treatment modality, which resulted in 90% survival if animals were treated 24 h after lethal ANDV challenge (B). Interestingly, the VSVΔG/ZEBOVGP vaccine resulted in a higher survival rate of 100% when administered 24 h postexposure and in 67% survival when administered 3 days postexposure (B). Although we have provided evidence for the role of neutralizing antibodies in the protection of hamsters immunized prophylactically (B and B), a pivotal role of the humoral immune response as the mechanism for postexposure treatment is unlikely. The innate immune response was thought to be, at least in part, essential for the success of the VSV platform in postexposure treatment for filovirus infections, keeping virus loads at levels that allow the adaptive immune response, which otherwise would be developed too late to clear the virus (12
). The fact that we see protection from the control vaccine (VSVΔG/ZEBOVGP) also supports the induction of a nonspecific innate immune response following immunization with VSV vectors, and this protection is similar to what we saw at 3 and 7 days postimmunization. This was not tested with or compared to wild-type VSV, because this virus causes an acute infection with a lethal outcome in the Syrian hamster model, even at very low doses (13
). The higher survival rates seen in the groups receiving VSVΔG/ZEBOVGP than in those receiving VSVΔG/ANDVGPC may be due either to the quicker replication of that virus () or to the significant difference in tropism of the glycoproteins. ZEBOV glycoproteins have a cell tropism primarily for macrophages, monocytes, and dendritic cells, which may be important in mounting an early immune response (61
), whereas the ANDV glycoproteins target primarily endothelial cells (63
). This is in agreement with our real-time RT-PCR hamster cytokine data, where VSVΔG/ZEBOVGP stimulated the innate immune system more quickly and robustly than VSVΔG/ANDVGPC (). However, the nonspecificity of protection differs from the nature of the protection seen against filovirus challenge in the nonhuman primate model, where a homologous VSV vaccine was required in order to achieve successful postexposure protection (12
). As an alternative theory, one could argue that the recombinant VSV vaccine might interfere with, and thus control, ANDV replication, allowing only subclinical or mild infections. A similar concept has been developed for other vaccines given simultaneously with the challenge virus, resulting in a subclinical infection that allows the development of a protective humoral immune response (48
), and this was detected in our studies (B). This, however, needs to be experimentally verified once proper reagents and tools for the study of immune responses other than antibodies in hamsters become available.
In summary, our studies have demonstrated the potential of a replication-competent VSV vector expressing the ANDV GPC for both prophylaxis and postexposure treatment of ANDV infection. Future studies will focus on further unraveling the mechanism of protection of this promising vaccine candidate.