Lethal Respiratory Challenge Research Articles

LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Epicutaneous immunization with modified vaccinia Ankara viral vectors generates superior T cell immunity against a respiratory viral challenge

Modified Vaccinia Ankara (MVA) was recently approved as a smallpox vaccine. Variola is transmitted by respiratory droplets and MVA immunization by skin scarification (s.s.) protected mice far more effectively against lethal respiratory challenge with vaccinia virus (VACV) than any other route of delivery, and at lower doses. Comparisons of s.s. with intradermal, subcutaneous, or intramuscular routes showed that MVAOVA s.s.-generated T cells were both more abundant and transcriptionally unique. MVAOVA s.s. produced greater numbers of lung Ova-specific CD8+ TRM and was superior in protecting mice against lethal VACVOVA respiratory challenge. Nearly as many lung TRM were generated with MVAOVA s.s. immunization compared to intra-tracheal immunization with MVAOVA and both routes vaccination protected mice against lethal pulmonary challenge with VACVOVA. Strikingly, MVAOVA s.s.-generated effector T cells exhibited overlapping gene transcriptional profiles to those generated via intra-tracheal immunization. Overall, our data suggest that heterologous MVA vectors immunized via s.s. are uniquely well-suited as vaccine vectors for respiratory pathogens, which may be relevant to COVID-19. In addition, MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine.

088 Skin delivery of modified vaccinia ankara (MVA) virus generates superior protective pulmonary immunity

No viral infection has shaped human history more profoundly than smallpox (Variola major). Although it was eradicated in vivo by a world-wide vaccination program by 1980, it remains a potential threat via bioterrorism, as most of the world’s population has never been immunized. There is an unacceptable incidence of morbidity after vaccinia virus (VACV) vaccination, particularly in individuals with atopic disorders. Therefore, a less virulent replication deficient variant of VACV, called MVA, has been developed. In all prior studies in humans, MVA has been delivered via intramuscular (im) injection. Variola major is transmitted by respiratory droplets, and successful immune response to V. major are T cell rather than antibody mediated. We showed that MVA as low as 1.8 X 106 pfu delivered by skin scarification (ss) elicited strong T cell mediated responses that completely protected mice against lethal respiratory challenge. MVA could be safely inoculated to skin of immunocompromised mice without any morbidity. Nearly equivalent numbers of lung tissue TRM could be generated via MVA ss compared to direct pulmonary infection; both were sufficient to protect mice against lethal pulmonary challenge with WT VACV. Strikingly, MVA ss-generated activated T cells exhibiting similar global gene transcriptional profile with those generated via pulmonary infection. Moreover, comparisons of ss with other vaccine delivery routes employed in clinic (id, sc or im) showed that MVA ss-generated T cells were both more abundant and qualitatively superior. Specifically, MVA ss produced more lung CD8+ TRM and was superior in protecting mice against lethal respiratory challenge. Mice genetically deficient in LC and Langerin+ DC demonstrated that both are required for generating an optimal CD8+ T cell response. Our data show that ss with MVA generates robust pulmonary protective immunity, and could be used as a safe and superior vaccine strategy against smallpox. This may be the preferred mode of delivery for MVA as a vaccine vector for other infectious and malignant diseases.

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia.

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

Single vector platform vaccine protects against lethal respiratory challenge with Tier 1 select agents of anthrax, plague, and tularemia

Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

Effector responses of cytotoxic CD8+ T cells generated by utilizing an FcR-targeting vaccine strategy in an intracellular bacterial infection model.

Abstract F. tularensis is a highly virulent (Category A) gram-negative intracellular mucosal pathogen. It is believed that while humoral immunity delays the progression of infection, cellular immunity is critical for protection. Consequently, by identifying a vaccine strategy that would generate a pathogen-specific, cell-mediated, cytotoxic immune response will be key for protection against intracellular bacterial pathogens such as F. tularensis, which has no FDA approved vaccine available. For this purpose, we have successfully developed an unadjuvanted vaccine delivery system, which targets Ag to FcγR on antigen presenting cells (APC). However, there is a significant lack of information regarding the role of FcR in the generation of mucosal immunity and more specifically the protective role played by cytotoxic CD8+ T cells. For this reason, we have utilized our F. tularensis FcR-targeting vaccine platform to analyze the generation of F. tularensis-specific, memory CD8+ T lymphocytes and their effector, cytotoxic functions during lethal respiratory challenge with the bacterium. In our studies, we have concluded that our FcR-targeting strategy generates antigen-specific effector memory CD8+ T cells that have significantly improved cytotoxic capabilities against macrophages infected by the bacterium and are capable to transfer protection in naïve recipients.

056 Skin scarification with modified vaccinia ankara (MVA) virus generates protective pulmonary immunity

No viral infection has shaped human history more profoundly than smallpox. Although it was eradicated by a world-wide vaccination program by 1980, several fundamental challenges remain. There is an unacceptable incidence of morbidity and mortality after vaccinia virus (VACV) vaccine, particularly in individuals with atopic disorders. Therefore, less virulent variants of VAVC have been sought and a replication deficient variants of VACV, modified vaccinia ankara (MVA), has been developed. In all prior studies in humans, MVA has been delivered via intramuscular (im) injection. In this study, we showed that MVA as low as 1.8 X 106 pfu delivered by skin scarification (ss) elicited strong cellular immune responses that completely protected mice against lethal respiratory challenge. MVA with dose of 1.8 X 106 pfu could be safely inoculated to scarified skin of immunocompromised mice without causing any morbidity. MVA ss generated activated T cells exhibiting similar global gene transcriptional profile with those generated via intratracheal infection. Nearly equivalent numbers of lung tissue resident memory T cells could be generated via MVA ss compared to direct pulmonary infection; the former was sufficient to protect mice against lethal pulmonary challenge with WT VACV. Moreover, comparisons of ss with other vaccine delivery routes employed in clinic (intradermal, subcutaneous or im) showed MVA ss generated T cells both quantitatively more abundant and qualitatively distinct. Furthermore, MVA ss produced more lung CD8+ TRM and was superior in protecting mice against lethal respiratory challenge. Overall, our data show that skin scarification with MVA generates sufficient pulmonary protective immunity, and could be used as a superior and safe vaccine strategy against smallpox and vaccine vector for other infectious and malignant diseases.

A Distinct Lung-Interstitium-Resident Memory CD8+ T Cell Subset Confers Enhanced Protection to Lower Respiratory Tract Infection

The nature and anatomic location of the protective memory CD8(+) Tcell subset induced by intranasal vaccination remain poorly understood. We developed a vaccination model to assess the anatomic location of protective memory CD8(+) Tcells and their role in lower airway infections. Memory CD8(+) Tcells elicited by local intranasal, but not systemic, vaccination with an engineered non-replicative CD8(+) Tcell-targeted antigen confer enhanced protection to a lethal respiratory viral challenge. This protection depends on a distinct CXCR3(LO) resident memory CD8(+) T (Trm) cell population that preferentially localizes to the pulmonary interstitium. Because they are positioned close to the mucosa, where infection occurs, interstitial Trm cells act before inflammation can recruit circulating memory CD8(+) Tcells into the lung tissue. This results in a local protective immune response as early as 1day post-infection. Hence, vaccine strategies that induce lung interstitial Trm cells may confer better protection against respiratory pathogens.

Human antibody specificities that confer protective immunity to poxvirus infections (VAC11P.1109)

Abstract Immunization with vaccinia virus (VACV) induces cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibodies (Abs). However, the specificity and breadth of protective human Abs to poxviruses are largely unknown. We used a highly-optimized human hybridoma technology to generate a panel of 90 anti-VACV monoclonal Abs from vaccinia-immunized subjects or from subjects who had a history of naturally-acquired monkeypox infection. Then, we assessed the neutralizing and protective capabilities of the Abs in vitro and in a lethal respiratory VACV challenge in a mouse model. A large fraction of Abs from the panel (>40%) possessed neutralizing activity against VACV, cowpox and/or monkeypox viruses. Antibodies to a variety of envelope proteins in VACV extracellular virions (A33, B5) or mature virions (L1, A27, D8, H3) contributed substantially to neutralizing activity in vitro. For protective immunity in vivo, L1, A27, A33 and B5 antibodies conferred levels of protection that were comparable to those induced by live viral vaccination, especially when used in combination. Such antibody cocktails have promise for use in as immunotherapeutics in humans.

In situ imaging unveiled location of protective endogenous CD8 T cell response in lung (MUC1P.916)

Abstract The anatomic distribution of protective endogenous memory CD8 T cell response in the lung—a common site of pathogen invasion—is poorly understood. We report that endogenous CD8 T cell memory installed by local pulmonary, but not systemic, vaccination resulted in faster viral control and enhanced protection to a lethal respiratory viral challenge in mice. Enhanced protection depended on elicitation of a distinct CXCR3LO resident CD8 T cell memory population (CD8 TRM) confined to the pulmonary interstitium. CD8 TRM sensed the pathogen and rapidly initiated a local immune response before inflammation-induced recruitment of lung vascular and/or circulating memory CD8 cells. Imaging of protective CD8 T cell response in lungs by exploiting in situ staining with fluorescent MHC class I tetramers coupled with Sheet Plane Illumination Microscopy (SPIM) platform uncovered that CD8 TRM are retained within immunologically correct locations, which include the delicate alveoli walls outside of the alveolar capillary network - the most vulnerable sites affected by lower airway infection. Such a location of memory CD8 T cell response may allow prompt surveillance of infected alveolar epithelium without need for the egress into the alveolar space. In sum, our study unveiled novel dimensional information of memory T cell response that localize to those anatomic sites of the lung that are critical for host defense.

Superior protection and distinct phenotype of pulmonary CD8+ T cells elicited by mucosal vaccination (MUC5P.863)

Abstract CD8-positive T cells (CTLs) confer protective immunity against many infectious diseases, and thus the development of safe and efficient CTL-targeted vaccination is of high clinical significance. Although many respiratory pathogens invade the host through the lung mucosa, most licensed human vaccines elicit limited and systemic T cell responses, rather than strong local pulmonary responses. We therefore wondered whether elicitation of pulmonary CTL response with microbe-free CTL-targeted subunit vaccine might benefit the host and protect against respiratory infections. We discovered superior protection of humanized mice from lethal respiratory challenge with vaccinia virus with intranasal, but not intra-peritoneal, vaccination. Critically, this superior protection did not correlate with the magnitude of the systemic CTL response but was dependent on in situ elicitation of robust pulmonary epitope-specific CTLs, which were able to limit viral replication and prevent disease. Global transcriptome analysis revealed that pulmonary CTLs elicited by vaccination possessed distinct phenotypic and functional properties when compared to CTLs that homed to the spleen. To extend this finding, we developed an approach for eliciting protective CTL responses that utilizes nanoparticle-based mucosal delivery of epitope-targeted vaccine formulated with an optimal adjuvant.

A Mucosal Subunit Vaccine Protects against Lethal Respiratory Infection with Francisella tularensis LVS

Francisella tularensis (FT) is a highly virulent pathogen for humans and other mammals. Severe morbidity and mortality is associated with respiratory FT infection and there are concerns about intentional dissemination of this organism. Therefore, FT has been designated a category A biothreat agent and there is a growing interest in the development of a protective vaccine. In the present study, we determine the protective potential of a subunit vaccine comprised of the FT heat shock protein DnaK and surface lipoprotein Tul4 against respiratory infection with the live vaccine strain (LVS) of FT in mice. First, we establish an optimal intranasal immunization regimen in C57BL/6 mice using recombinant DnaK or Tul4 together with the adjuvant GPI-0100. The individual immunization regimens induced robust salivary IgA, and vaginal and bronchoalveolar IgA and IgG antigen-specific antibodies. Serum IgG1 and IgG2c antibody responses were also induced, indicative of a mixed type 2 and type 1 response, respectively. Next, we show that immunization with DnaK and Tul4 induces mucosal and systemic antibody responses that are comparable to that seen following immunization with each antigen alone. This immunization regimen also induced IFN-γ, IL-10 and IL-17A production by splenic CD4+ T cells in an antigen-specific manner. Importantly, over 80% of the mice immunized with DnaK and Tul4, but not with each antigen alone, were protected against a lethal respiratory challenge with FT LVS. Protection correlated with reduced bacterial burden in the lung, liver and spleen of mice. This study demonstrates the potential of DnaK and Tul4 as protective antigens and lends support to the notion of combining distinct, immunodominant antigens into an effective multivalent tularemia vaccine.

Controlled Inflammatory Responses in the Lungs Are Associated with Protection Elicited by a Pneumococcal Surface Protein A-Based Vaccine against a Lethal Respiratory Challenge with Streptococcus pneumoniae in Mice

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.

Protection against Lethal Vaccinia Virus Challenge by Using an Attenuated Matrix Protein Mutant Vesicular Stomatitis Virus Vaccine Vector Expressing Poxvirus Antigens

Recombinant vesicular stomatitis viruses (VSV) are excellent candidate vectors for vaccination against human diseases. The neurovirulence of VSV in animal models requires the attenuation of the virus for use in humans. Previous efforts have focused on attenuating virus replication. Studies presented here test an alternative approach for attenuation that uses a matrix (M) protein mutant (rM51R) VSV as a vaccine vector against respiratory infection. This mutant is attenuated for viral virulence by its inability to suppress the innate immune response. The ability of rM51R VSV vectors to protect against lethal respiratory challenge was tested using a vaccinia virus intranasal challenge model. Mice immunized intranasally with rM51R vectors expressing vaccinia virus antigens B5R and L1R were protected against lethal vaccinia virus challenge. A single immunization with the vectors provided protection against vaccinia virus-induced mortality; however, a prime-boost strategy reduced the severity of the vaccinia virus-induced disease progression. Antibody titers measured after the prime and boost were low despite complete protection against lethal challenge. However, immunized animals had higher antibody titers during the challenge, suggesting that memory B-cell responses may be important for the protection. Depletion experiments demonstrated that B cells but not CD8 T cells were involved in the protection mediated by rM51R vaccine vectors that express B5R and L1R. These results demonstrate the potential of M protein mutant VSVs as candidate vaccine vectors against human diseases.

Epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective T cell-mediated immunity.

Introductory ParagraphVariola major infection (Smallpox) claimed hundreds of millions lives before it was eradicated by a simple vaccination strategy: epicutaneous application of the related orthopoxvirus Vaccinia virus (VACV) to superficially injured skin (skin scarification, s.s.)1. However, the remarkable success of this strategy was attributed to the immunogenicity of VACV rather than the unique vaccine delivery mode. We now demonstrate that VACV immunization via s.s., but not conventional injection routes, is essential to the generation of superior T cell-mediated immune responses that provide complete protection against subsequent challenges, independent of neutralizing antibodies. Skin-resident effector memory T cells (TEM) provide complete protection against cutaneous challenge, while protection against lethal respiratory challenge requires both respiratory mucosal TEM and central memory T cells (TCM). Vaccination with recombinant VACV (rVACV) expressing a tumor antigen was protective against tumor challenge only if delivered via s.s. route, but not by hypodermic injection. Finally, the clinically safer non-replicative Modified Vaccinia Ankara (MVA) also generated far superior protective immunity when delivered via s.s route compared to intramuscular injection used in MVA clinical trials. Thus, delivering rVACV -based vaccines, including MVA vaccines, through physically disrupted epidermis represents a uniquely powerful strategy with clear-cut advantages over conventional vaccination via hypodermic injection.

Expression of B-Cell Activating Factor Enhances Protective Immunity of a Vaccine againstPseudomonas aeruginosa

B-cell activating factor (BAFF), a member of the TNF family, is a potent cytokine with stimulatory effects on B and T cells. To evaluate the potential of transient overexpression of BAFF to enhance vaccine immunogenicity, a replication-deficient adenovirus expressing full-length murine BAFF (AdBAFF) was tested in a mouse vaccine model against Pseudomonas aeruginosa. When coadministered with heat-killed P. aeruginosa, AdBAFF mediated a significant increase in anti-P. aeruginosa-specific serum and lung mucosal antibodies and resulted in improved protection against a lethal respiratory challenge with P. aeruginosa. This effect was independent of the site of administration of AdBAFF and was observed both when AdBAFF was given simultaneously with heat-killed P. aeruginosa as well as when AdBAFF was administered 4 weeks after immunization with heat-killed P. aeruginosa. These data demonstrate that a temporal increase in systemic BAFF levels is able to augment a P. aeruginosa-specific immune response upon immunization with heat-killed P. aeruginosa, suggesting that the immune-stimulatory effects of BAFF may be exploited as a molecular adjuvant for genetic vaccines.

Structural Basis for Potent Cross-Neutralizing Human Monoclonal Antibody Protection against Lethal Human and Zoonotic Severe Acute Respiratory Syndrome Coronavirus Challenge

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants.

Structural Basis for Potent Cross-Neutralizing Human Monoclonal Antibody Protection against Lethal Human and Zoonotic Severe Acute Respiratory Syndrome Coronavirus Challenge

Structural Basis for Potent Cross-Neutralizing Human Monoclonal Antibody Protection against Lethal Human and Zoonotic Severe Acute Respiratory Syndrome Coronavirus Challenge

Immune Serum Produced by DNA Vaccination Protects Hamsters against Lethal Respiratory Challenge with Andes Virus

Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus Hantavirus of the family Bunyaviridae. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD(50)], approximately 100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days -1 and +5 relative to challenge were protected against intranasal challenge (21 LD(50)). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.

Tail scarification with Vaccinia virus Lister as a model for evaluation of smallpox vaccine potency in mice

Since smallpox eradication by the WHO during the 1980s, potency of new vaccines is compared to vaccines that were used during the eradication campaign. In this work we characterize the tail scarification technique in mice as a model for scarification in humans. Similar to humans, mice develop “clinical take” which is dependent on the vaccination dose. Appearance of anti-Vaccinia IgM is followed by IgG antibodies 10 days post scarification and lasting more then 1½ years. Mice with “clinical take” are 100% protected against lethal respiratory challenge (100 LD 50) of Vaccinia WR indicating that the “clinical take” can serve as a correlate of protective immunity. Reducing the vaccination dose and using Cowpox virus as a more virulent strain, enabled us to draw the limit of the vaccine potency in mice. Similar to humans, in revaccinated mice the development of “clinical take” was inversely correlated to the level of pre-existing antibodies. These results indicate that tail scarification of mice can be used as a model for evaluation of smallpox vaccines. High correlation between “clinical take” and protective immunity allows the use of visual inspection to evaluate vaccine potency.