JOURNAL OF VIROLOGY, Sept. 2008, p. 9273–9277 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.00915-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 18
Immune Response in the Absence of Neurovirulence in Mice Infected with M Protein Mutant Vesicular Stomatitis Virus䌤 Maryam Ahmed,1* Tracie R. Marino,1 Shelby Puckett,1 Nancy D. Kock,2 and Douglas S. Lyles1 Departments of Biochemistry1 and Pathology/Comparative Medicine,2 Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 Received 2 May 2008/Accepted 19 June 2008
Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors. containing the M51R M protein mutation (25). These results suggest that the suppression of host innate immune responses by M protein is a major determinant of virulence for VSV and that rM51R-M virus would be a safer and more effective vaccine vector than wt VSV strains. Although we and others have shown that M protein mutants of VSV are attenuated for spread in the central nervous system (CNS) (1, 25), it is not known at what step during its progression to the CNS the virus is cleared. In addition, antibody responses to M protein mutants have not been analyzed in detail. In this study, we sought to investigate whether rM51R-M virus exhibits the properties of an effective vaccine vector by determining its ability to spread in the CNS and also induce an effective antibody response in vivo. rM51R-M virus is attenuated for spread to the CNS. Previous studies have demonstrated that when wt VSV is inoculated intranasally in mice, its initial replication occurs in respiratory and olfactory epithelial cells. This is followed by neuronal spread through the olfactory tract to the CNS, resulting in viral encephalitis (10, 11). To determine the ability of rM51R-M virus to replicate and spread to the CNS, 7- to 8-week-old female C57BL/6 mice were inoculated by the intranasal route with 1 ⫻ 107 PFU of virus. Mice infected with the rwt virus served as positive controls in this experiment. At days 1, 2, 4, and 7, the animals were sacrificed, and the whole heads, lungs, and spleens were harvested and preserved in 4% paraformaldehyde. Following fixation, the heads were decalcified, and horizontal sections of the head, including sections through the nose and brain, and the soft tissues were trimmed appropriately. All tissues were embedded in paraffin, sectioned, and stained for immunohistochemical analysis with antibodies against the viral G protein and/or with hematoxylin and eosin. Images of the nasal passages demonstrate strong positive immunohistochemical staining of the nasal epithelium in the VSV-infected animals (Fig. 1). At day 1 postinfection, there was strong diffuse staining of nasal mucosal epithelial cells in both the rwt and rM51R-M virus-infected mice. Viral antigen was also detected in the rwt virus-infected mice at days 2 and 4 postinfection, but staining for the G protein was negative in the noses of rM51R-M virus-infected mice by day 2. As ex-
Recombinant vesicular stomatitis virus (VSV)-based vectors expressing foreign proteins are currently being explored as vaccines due to their ability to induce strong immune responses and protect against challenges with a variety of pathogens, including human immunodeficiency virus and influenza virus (7, 12, 19–21). Although laboratory-adapted strains of VSV are generally nonpathogenic in humans and nonhuman primates, the potential for VSV vectors to cause disease in humans has been addressed by the development of vectors that are attenuated for virus growth by deleting or mutating the viral glycoprotein (G protein) or by rearranging the natural gene order of VSV (4, 5, 8, 18). In many cases, this has the undesired effect of reducing the level of antigen expression and reducing the subsequent immune response. An alternative strategy is to attenuate viral pathogenicity by reducing the ability of the virus to suppress host innate immune responses without compromising the yield of infectious progeny. This strategy has the advantages of high levels of antigen expression and also the enhancement of adaptive immune responses through the activation of innate immune responses. Wild-type (wt) strains of VSV effectively suppress the host innate immune response through the inhibition of host gene expression by the viral matrix (M) protein (2, 23). M protein is a multifunctional protein that is involved in the shutoff of host transcription, nuclear cytoplasmic transport, and translation during virus infection (13). Studies previously carried out in our laboratory demonstrate that a recombinant M protein mutant of VSV, rM51R-M virus, containing an arginine for methionine substitution at position 51 of the M protein sequence, is defective at inhibiting host gene expression (2). Therefore, in contrast to its isogenic recombinant wt counterpart (rwt virus), rM51R-M virus stimulates the expression of genes involved in host innate immune responses. Furthermore, rM51R-M virus does not cause disease in mice (1), nor do other strains of VSV
* Corresponding author. Mailing address: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Phone: (336) 716-1589. Fax: (336) 716-7176. E-mail:
[email protected]. 䌤 Published ahead of print on 9 July 2008. 9273
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FIG. 2. Hematoxylin- and eosin-stained histological sections of the nasal passages of VSV-infected mice at days 1, 2, and 4 (D1, D2, and D4, respectively) after infection with the rwt and rM51R-M viruses. Magnification, ⫻4. Magnification of inset images, ⫻40. Mild inflammation with hemorrhages and a few inflammatory cells are present in both at day 1. The injury progresses with the rwt virus from days 2 to 4, while there are no significant lesions with the rM51R-M virus by day 2. This further injury is characterized by mucosal ulceration, with epithelial sloughing and more intense luminal inflammation.
FIG. 1. Immunohistochemical staining for VSV antigens in the nasal passages and brains of mice. Magnification, ⫻4. Images of the nasal passages of rwt and rM51R-M virus-infected animals at days 1, 2, 4, and 7 (D1, D2, D4, and D7, respectively) are shown in the top four rows of images. The images in the bottom row show the olfactory bulb region of the brain at day 7 postinfection (D7-OB). The inset images are shown at ⫻40 magnification. In the positive images (those with red staining), the epithelial surfaces of the nasal cavity show strong cytoplasmic and mucosal surface immunoreactivity. Similar staining is present in the olfactory bulb at day 7 with the rwt virus.
pected, the rwt virus had spread to the olfactory bulb of the brain by day 7, while the rM51R-M virus was undetectable at that site. These data are consistent with an earlier analysis of the infectious virus by plaque assay (1). These results indicate that the rM51R-M virus is attenuated for growth and spread in the CNS and is cleared from nasal mucosa by day 2 postinfection. Figure 2 shows hematoxylin- and eosin-stained nasal tissues at different times postinfection. At day 1, the mucosal surfaces were intact in both the rwt and rM51R-M virus-infected mice, and small numbers of erythrocytes and inflammatory cells (primarily neutrophils) were present in the lumina. However, by day 2 postinfection, erosions of the mucosal surface were present in the rwt virus-infected animals, and by day 4, they had progressed to ulcers with increasing submucosal and luminal inflammation (Fig. 2, insets). In contrast, the mucosal surfaces in the mice infected with rM51R-M virus were normal at these time points, and exudate was not found in the lumina,
further supporting the low virulence of the rM51R-M virus in vivo. The presence of viral antigen in the lungs and spleens of infected mice was analyzed to determine the spread of virus to peripheral tissues (Fig. 3). We were able to detect low levels of viral antigen in the lungs of both the rwt and rM51R-M virusinfected mice at day 4 (Fig. 3A). Although viral antigen-positive cells were also observed in the rwt virus-infected animals at day 7, we were unable to detect viral antigen in the lungs of mice infected with rM51R-M virus at that time. For both the rwt and rM51R-M viruses, viral titers in the lungs were below the level of detection at any time point tested (days 1 to 7). A previous study reported the presence of infectious virus in the lungs of mice infected with wt VSV as well as an M51 deletion mutant (16). Differences from our study may be due to differences in virus strains or in our inoculation technique, which avoided the delivery of the inoculum directly to the lung. Viral antigen-positive cells were detected in the spleens of mice infected with the rwt and rM51R-M viruses at days 4 and 7 postinfection (Fig. 3B). However, in some cases, VSV antigen-positive cells were observed at day 2 (data not shown). Most of the staining was restricted to cells in the red pulp of the spleen, which consists of circulating red blood cells and immune cells. However, in the rwt virus-infected mice, viral antigen-positive staining was occasionally detected in the white pulp of the spleen (Fig. 3B, rwt virus, day 7), suggesting the stimulation of immune cells in response to the wt virus. As observed for the lungs, infectious virus was not detected in the spleens of mice infected with either virus from days 1 to 7 postinfection. Similar experiments with wt VSV have demon-
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FIG. 3. Immunohistochemical staining for VSV antigens in the lungs (A) and spleens (B) of mice 4 and 7 days postinfection (D4 and D7, respectively) with the rwt and rM51R-M viruses. Magnification, ⫻60. Positive cells (those with red staining) are scattered throughout both organs with the rwt virus and in all but the lungs at day 7 with the rM51R-M virus.
strated the presence of cells containing VSV antigen or genome in the spleens of mice in the absence of infectious virus for several months (22, 26). Pathological changes were not detected in the spleens or lungs with either of the viruses following intranasal infection. This is in contrast to the dramatic inflammatory response observed in the noses and CNS of the rwt virus-infected mice. Although we cannot rule out the possibility that a low level of infectious virus was present at those sites, it is likely that the systemic spread of virus to peripheral organs induced an effective immune response that limited virus replication and pathogenicity at those sites. Furthermore, although the rM51R-M virus was attenuated for spread through the nasal passages and did not cause disease in vivo, our results suggest that it also has the potential to elicit an effective anti-VSV immune response, as indicated by the presence of viral antigen-positive cells in the spleen. Antibody titers in mice infected with rM51R-M virus are comparable to those obtained from rwt virus-infected mice. To determine the antibody response to rM51R-M virus, mice were intranasally infected with different doses of rM51R-M virus (Fig. 4). As a positive control for the elicitation of an effective antibody response, mice were infected with the rwt virus at 1 ⫻ 104 (Fig. 4A and B) or 1 ⫻ 105 PFU (Fig. 4C and D), which is near the 50% lethal dose for intranasal inoculation (1). Blood was collected on day 7, 14, 21, or 28 postinfection, and VSVspecific antibody titers were determined by enzyme-linked immunosorbent assay. The titers did not change significantly after day 14 postinfection. Mice infected with rM51R-M virus at 1 ⫻ 104 and 1 ⫻ 105 PFU had low total antibody titers (Fig. 4A). However, when infected at 1 ⫻ 107 PFU, all animals had
FIG. 4. (A) Anti-VSV antibody titers in mice infected with the rM51R-M or rwt viruses. Mice were infected intranasally with the indicated doses of the rM51R-M or rwt viruses. Total serum Ig titers were determined by enzyme-linked immunosorbent assay (ELISA) using purified VSV as antigen. The symbols represent the titers of individual mice at 14 days postinfection, the bars represent geometric mean titers, and the numbers in parentheses represent the number of mice/total with titers above the negative control. (B) Weight change in mice infected with the rM51R-M or rwt virus. The data shown are the mean weights of the mice described in panel A infected with the rM51R-M virus at 104 PFU (open squares), 105 PFU (open circles), or 107 PFU (open triangles) or the rwt virus at 104 PFU (filled squares). (C and D) Anti-VSV IgG1 and IgG2a titers of mice infected with the rM51R-M or rwt virus. The data shown are the geometric means ⫾ standard deviations of the results for five mice per group from a separate experiment from the one described in panel A.
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seroconverted, and antibody levels were similar to those observed in the rwt virus-infected mice. Although their antibody titers were similar, mice infected with the rwt virus showed signs of VSV-induced disease, as indicated by a statistically significant weight loss beginning on day 7 postinfection compared to the mice infected with rM51R-M virus (Fig. 4B). None of the mice infected with rM51R-M virus showed any clinical signs of illness throughout the course of the experiment (28 days). Immunoglobulin G1 (IgG1) and IgG2a titers in mice infected with 1 ⫻ 107 PFU of rM51R-M virus were comparable to those obtained in mice infected with the rwt virus (Fig. 4C and D, respectively). For both viruses, the IgG2a response was approximately 1 log higher than the IgG1 response. These results indicate that the isotype distribution of the rM51R-M virus-infected animals was similar to that obtained with the rwt positive control. The experiments presented here show that rM51R-M virus was more rapidly cleared from the nasal mucosa than the rwt virus and was attenuated for spread in the CNS, thus accounting for its reduced virulence. However, it did induce an antibody response similar to that of the mice infected with wt VSV, most likely due to the systemic spread of virus to peripheral organs, such as the spleen and lymph nodes. Previous studies have shown that wt VSV infection of immunocompetent mice results in the CD4⫹ T-cell-dependent production of neutralizing IgG antibodies and the induction of a virus-specific cytotoxic T lymphocyte response (9, 14, 17). Therefore, given the similarity in the antibody responses induced by the rwt and rM51R-M viruses, it is likely that rM51R-M virus induces T-cell and memory responses comparable to those observed with wt VSV strains. Our strategy to develop an effective VSV vaccine vector for the delivery of foreign antigens was to reduce the ability of the virus to suppress host innate immune responses without compromising the yield of infectious progeny. Similar approaches have been developed with other viruses by introducing mutations in genes whose products suppress host antiviral responses. For example, influenza viruses with deletions in the NS1 gene are less pathogenic than wt strains but elicit strong adaptive immune responses (6, 15, 24). In addition, interferoninducing mutants of bovine respiratory syncytial virus lacking the NS proteins (27) and simian virus 5 with P/V mutations (3) are also being developed as vaccine vectors. Alternative strategies for reducing the pathogenicity associated with VSV infection have focused on reducing the ability of the virus to produce infectious progeny by deleting or mutating the viral G protein (4, 18) or by rearranging the natural gene order of VSV (4, 8). Although these alterations resulted in decreased neurovirulence, the efficacy of these vectors was reduced due to a decrease in the level of antigen expression. However, vectors designed using combinations of attenuation phenotypes have had more success, not only in effectively reducing neurovirulence but also in retaining immunogenicity (5). Although it is likely that such combination approaches will be important for the development of effective vaccine vectors, the results presented here show that rM51R-M virus induced strong immunity in vivo without causing disease, thus demonstrating the potential of viruses that stimulate innate immunity as successful delivery vehicles for target antigens. Future studies
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