Enhancement of immune response to an antigen delivered by vaccinia virus by displaying the antigen on the surface of intracellular mature virion

Vaccinia virus (VACV) is the vaccine for smallpox and a widely used vaccine vector for infectious diseases and cancers. The majority of the antibodies elicited by live VACV vaccination respond to virion structural proteins, including many integral membrane proteins on the intracellular mature virion (MV). Here, we showed that antibody response to an exogenous antigen delivered by VACV was greatly enhanced by incorporating the antigen as an integral membrane protein of MV. We constructed recombinant VACV expressing a Yersinia pestis protective antigen, LcrV, unmodified or fused with either a signal peptide or with the transmembrane domain of VACV D8 protein (LcrV-TM). Electron microscopy showed that LcrV-TM was displayed on the surface of MV. Importantly, VACV expressing LcrV-TM elicited a significantly higher titer of anti-LcrV antibody in mice than viruses expressing other forms of LcrV. Only mice immunized with LcrV-TM-expressing VACV were protected from lethal Y. pestis and VACV WR challenges. Antigen engineering through fusion with D8 transmembrane domain may be broadly applicable for enhancing the immune response to antigens delivered by a VACV vector. The recombinant virus described here could also serve as the basis for developing a vaccine against both smallpox and plague.     

Vaccinia viruses with mutations in the E3L gene as potential replication-competent, attenuated vaccines: intra-nasal vaccination

Vaccinia virus (VACV) has been used as the vaccine to protect against smallpox, and recombinant VACVs have been used to develop vaccine candidates against numerous cancers and infectious diseases. Although relatively safe for use in humans, the strains of VACV that were used as smallpox vaccines led to several complications including, progressive infection in immune compromised individuals, eczema vaccination in individuals with a history of atopic dermatitis, and encephalitis and perimyocarditis in apparently healthy individuals. The work described in this paper focuses on attenuated strains of VACV that may have the potential for use as vaccine vectors with reduced pathogenicity. We have generated several VACV mutants in a WR background with specific mutations in the E3L gene that were at least a 1000-fold less pathogenic compared to wtVACV upon intra-nasal infection of mice. Many of these mutant viruses replicated to high titers in the nasal mucosa of mice following intra-nasal administration. Despite replication to high titers in the nose, there was little spread to other organs in infected animals. Intra-nasal vaccination with doses as low as 100-1000 pfu (plaque forming units) of these replicating VACV constructs were sufficient to protect the host from challenge with large doses of wtVACV. Similar constructs in a Copenhagen and a NYCBH background were highly attenuated, yet effective as vaccines in the mouse model. These recombinant VACV constructs may be promising vector candidates for use in vaccination strategies against smallpox and other pathogens.     

Genomic differences of Vaccinia virus clones from Dryvax smallpox vaccine: the Dryvax-like ACAM2000 and the mouse neurovirulent Clone-3

Conventional vaccines used for smallpox eradication were often denoted one or another strain of Vaccinia virus (VACV), even though seed virus was sub-cultured multifariously, which rendered the virion population genetically heterogeneous. ACAM2000 cell culture vaccine, recently licensed in the U.S., consists of a biologically vaccine-like VACV homogeneous-sequence clone from the conventional smallpox vaccine Dryvax, which we verified from Dryvax sequence chromatograms is genetically heterogeneous. ACAM2000 VACV and CL3, a mouse-neurovirulent clone from Dryvax, differ by 572 single nucleotide polymorphisms and 53 insertions–deletions of varied size, including a 4.5-kbp deletion in ACAM2000 and a 6.2-kbp deletion in CL3. The sequence diversity between the two clones precludes precisely defining why CL3 is more pathogenic; however, four genes appear significantly dissimilar to account for virulence differences. CL3 encodes intact immunomodulators interferon-/β and tumor necrosis factor receptors, which are truncated in ACAM2000. CL3 specifies a Cowpox and Variola virus-like ankyrin-repeat protein that might be associated with proteolysis via ubiquitination. And, CL3 shows an elongated thymidylate kinase, similar to the enzyme of the mouse-neurovirulent VACV-WR, a derivative of the New York City Board of Health vaccine, the origin vaccine of Dryvax. Although ACAM2000 encodes most proteins associated with immunization protection, the cloning probably delimited the variant epitopes and other motifs produced by Dryvax due to its VACV genetic heterogeneity. The sequence information for ACAM2000 and CL3 could be significant for resolving the dynamics of their different proteomes and thereby aid development of safer, more effective vaccines.     

Laboratory-acquired vaccinia exposures and infections--United States, 2005-2007

The last case of naturally acquired smallpox disease, caused by the orthopoxvirus variola virus (VARV), occurred in 1977, and the last laboratory-acquired case occurred in 1978. Smallpox was eradicated largely as the result of a worldwide vaccination campaign that used the related orthopoxvirus, vaccinia virus (VACV), as a live virus vaccine. Routine childhood vaccination for smallpox in the United States was terminated by 1972, but vaccination continues or has been reintroduced for specific groups, including laboratory workers who may be exposed to orthopoxviruses, members of the military, selected health-care workers, and first responders. Severe complications of VACV infection can occur, particularly in persons with underlying risk factors, and secondary transmission of VACV also can occur. VACV is used in numerous institutions for various research purposes, including fundamental studies of orthopoxviruses and use as a vector for the expression of foreign proteins (often antigens or immunomodulators) in eukaryotic cells and animal models. The widespread use of VACV for research has resulted in laboratory-acquired VACV infections, some requiring hospitalization. The current Advisory Committee on Immunization Practices (ACIP) guidelines recommend VACV vaccination for laboratory workers who handle cultures or animals contaminated or infected with nonhighly attenuated VACV strains or other orthopoxviruses that infect humans. This report describes five recent occurrences of laboratory-acquired VACV infections and exposure and underscores the need for proper vaccination, laboratory safety, infection-control practices, and rapid medical evaluation of exposures in the context of orthopoxvirus research.     

Smallpox vaccine,ACAM2000: Sites and duration of viral shedding and effect of povidone iodine on scarification site shedding and immune response

The U.S. Department of Defense vaccinates personnel deployed to high-risk areas with the vaccinia virus (VACV)-based smallpox vaccine. Autoinoculations and secondary and tertiary transmissions due to VACV shedding from the vaccination site continue to occur despite education of vaccinees on the risks of such infections. The objectives of this study were to investigate, in naïve smallpox vaccinees, (a) whether the vaccination site can remain contagious after the scab separates and (b) whether the application of povidone iodine ointment (PIO) to the vaccination site inactivates VACV without affecting the immune response. These objectives were tested in 60 individuals scheduled to receive smallpox vaccine. Thirty individuals (control) did not receive PIO; 30 subjects (treatment) received PIO starting on post-vaccination day 7. Counter to current dogma, this study showed that VACV continues to shed from the vaccination site after the scab separates. Overall viral shedding levels in the PIO group were significantly lower than those in the control group (p = 0.0045), and PIO significantly reduced the duration of viral shedding (median duration 14.5 days and 21 days in the PIO and control groups, respectively; p = 0.0444). At least 10% of control subjects continued to shed VACV at day 28, and 3.4% continued to shed the virus at day 42. PIO reduced the proportion of subjects shedding virus from the vaccination site from day 8 until days 21–23 compared with control subjects. Groups did not differ significantly in the proportion of subjects mounting an immune response, as measured by neutralizing antibodies, IgM, IgG, and interferon-gamma enzyme-linked immunospot assay. When applied to the vaccination site starting on day 7, PIO reduced viral shedding without altering the immune response. The use of PIO in addition to a semipermeable dressing may reduce the rates of autoinoculation and contact transmission originating from the vaccination site in smallpox-vaccinated individuals.     

Differential efficacy of vaccinia virus envelope proteins administered by DNA immunisation in protection of BALB/c mice from a lethal intranasal poxvirus challenge

DNA vaccines might offer an alternative to the live smallpox vaccine in providing protective efficacy in an orthopoxvirus (OPV) lethal respiratory challenge model. BALB/c mice were immunised with DNA vaccines coding for 10 different single vaccinia virus (VACV) membrane proteins. After an intranasal challenge with the VACV IHD strain, three gene candidates B5R, A33R and A27L produced > or =66% survival. The B5R DNA vaccine consistently produced 100% protection and exhibited greatest efficacy after three 50 microg intramuscular doses in this model. Sero-conversion to these vaccines was often inconsistent, implying that antibody itself was not a correlate of protection. The B5R DNA vaccine induced a strong and consistent gamma interferon (IFNgamma) response in BALB/c mice given a single DNA vaccine dose. Strong IFNgamma responses were also measured in pTB5R immunised C57BL6 mice deficient for MHC class I molecules, suggesting that the memory response was mediated by a CD4+ T cell population.     

Vaccination with a codon-optimized A27L-containing plasmid decreases virus replication and dissemination after vaccinia virus challenge

Smallpox is a disease caused by Variola virus (VARV). Although eradicated by WHO in 1980, the threat of using VARV on a bioterror attack has increased. The current smallpox vaccine ACAM2000, which consists of live vaccinia virus (VACV), causes complications in individuals with a compromised immune system or with previously reported skin diseases. Thus, a safer and efficacious vaccine needs to be developed. Previously, we reported that our virus-free DNA vaccine formulation, a pVAX1 plasmid encoding codon-optimized VACV A27L gene (pA27LOPT) with and without Imiquimod adjuvant, stimulates A27L-specific production of IFN-γ and increases humoral immunity 7 days post-vaccination. Here, we investigated the immune response of our novel vaccine by measuring the frequency of splenocytes producing IFN-γ by ELISPOT, the TH1 and TH2 cytokine profiles, and humoral immune responses two weeks post-vaccination, when animals were challenged with VACV. In all assays, the A27-based DNA vaccine conferred protective immune responses. Specifically, two weeks after vaccination, mice were challenged intranasally with vaccinia virus, and viral titers in mouse lungs and ovaries were significantly lower in groups immunized with pA27LOPT and pA27LOPT + Imiquimod. These results demonstrate that our vaccine formulation decreases viral replication and dissemination in a virus-free DNA vaccine platform, and provides an alternative towards a safer an efficacious vaccine.     

Comparative efficacy of replicating smallpox vaccine strains in a murine challenge model

There is currently considerable concern about the vulnerability of human populations to biowarfare or bioterrorist attacks with variola virus (VARV). Traditional smallpox vaccines were manufactured using the lymph of ruminants infected with the vaccinia virus (VACV). However, these production methods do not meet current standards for vaccines, especially since the emergence of transmissable spongiform encephalopathies in domesticated ruminants. This study has examined the protective efficacy of the Lister (Elstree) vaccine strain from various sources in a murine lethal challenge model. Considerable variation in efficacy is observed between the Lister material obtained from the American Type Culture Collection (ATCC) and the same strain obtained from vaccine stockpiles. A new, tissue-culture derived Lister vaccine is assessed against a bench-mark of multiple lots from a historical stockpile of the traditional vaccine. Apparent qualitative differences are observed between historical and new vaccines. Statistically significant differences are observed between different batches of the traditional vaccine, and the efficacy of the tissue-culture produced vaccine falls within this range.     

Diverse recognition of conserved orthopoxvirus CD8+ T cell epitopes in vaccinated rhesus macaques

Vaccinia virus (VACV) induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. To identify immunodominant poxvirus proteins and to facilitate future testing of smallpox vaccines in non-human primates, we used an algorithm for the prediction of VACV peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 VACV ORFs; 100 of these peptides did not contain the canonical proline at position three of the Mamu-A*01 binding motif. Cellular immune responses in PBMC from two vaccinia-vaccinated Mamu-A*01+ macaques were assessed by IFNγ ELISPOT assays. Vaccinated macaques recognized 17 peptides from 16 different ORFs with 6 peptides recognized by both macaques. Comparison with other orthopoxvirus sequences revealed that 12 of these epitopes are strictly conserved between VACV, variola, and monkeypoxvirus. ELISPOT responses were also observed to eight epitopes that did not contain the canonical P3 proline. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple VACV proteins and that a subset of these T cell epitopes is highly conserved among orthopoxviruses.     

Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies

Modified Vaccinia virus Ankara (MVA) is a tissue culture-derived, highly attenuated strain of vaccinia virus (VACV) exhibiting characteristic defective replication in cells from mammalian hosts. In the 1960s MVA was originally generated as a candidate virus for safer vaccination against smallpox. Now, MVA is widely used in experimental vaccine development targeting important infectious diseases and cancer. Versatile technologies for genetic engineering, large-scale production, and quality control facilitate R&D of recombinant and non-recombinant MVA vaccines matching today's requirements for new biomedical products. Such vaccines are attractive candidates for delivering antigens from pathogens against which no, or no effective vaccine is available, including emerging infections caused by highly pathogenic influenza viruses, chikungunya virus, West Nile virus or zoonotic orthopoxviruses. Other directions are seeking valuable vaccines against highly complex diseases such as AIDS, malaria, and tuberculosis. Here, we highlight examples of MVA candidate vaccines against infectious diseases, and review the efforts made to assess both the efficacy of vaccination and immune correlates of protection in preclinical studies.     

Brazilian vaccinia viruses and their origins

Although the World Health Organization (WHO) declared global smallpox eradicated in 1980, concerns over emergent poxvirus infections have increased. Most poxvirus infections are zoonotic; exploring their genetic diversity will illuminate the genetic and evolutionary aspects of poxvirus infections, ecology, and epidemiology. In recent decades, several strains of the orthopoxvirus vaccinia virus (VACV) have been isolated throughout Brazil, including genetically distinct isolates within the same outbreak. To further investigate the diversity and origins of these viruses, we analyzed molecular data from 8 Brazilian VACV isolates and compared several genes involved in virus structure and pathogenicity. Genetic variation among isolates suggests that ancestral Brazilian VACVs existed before the beginning of the WHO smallpox eradication vaccination campaigns and that these viruses continue to circulate.     

Mouse neurotoxicity test for vaccinia-based smallpox vaccines

The only US FDA licensed smallpox vaccine, Dryvax, was associated with rare but serious neurological adverse events. After smallpox was eradicated in the United States, mass vaccination ceased in 1971. As counter-bioterrorism/biowarfare measures, new smallpox vaccines are now being investigated. However, there are no established pre-clinical neurotoxicity assays with which to evaluate these new vaccines prior to licensure. Here we report the development and initial characterization of a small animal neurotoxicity assay for vaccinia-based smallpox vaccines using Dryvax virus as a reference vaccine strain and the neuroadapted Western Reserve (WR) strain as a neurotoxic positive control. In neonatally inoculated mice, the WR strain produced significantly greater and more rapid onset of mortality than the Dryvax vaccine reference. Expression of virus antigen in neural cells and infectious virus replication in the brain was also significantly different between the two strains. In addition, the appearance of high titer virus antibody correlated with the clearance of virus from brain. With further validation, this assay incorporating a licensed vaccine reference standard and positive control strain may provide important pre-clinical neurotoxicity data on new vaccinia-based smallpox vaccine strains.     

Genetically engineered poxviruses: a novel approach to the construction of live vaccines

Gene splicing techniques have been used to modify the smallpox vaccine virus thus providing a generic approach for the construction of live vaccines directed against a variety of heterologous infectious disease agents. The technique involves translocating a particular gene from an infectious agent into the genetic material of the smallpox vaccine virus. This unique foreign gene, selected because it contains the information essential for the synthesis of an antigen important in immunity to that particular infectious disease agent, is now expressed under the regulation of the engineered smallpox vaccine virus. On immunization with this live recombinant vaccine, the body is fooled into thinking that it was infected by the foreign infectious disease agent and mounts a defensive attack resulting in immunity to that particular infectious agent. Three examples of this approach are provided. Thus, smallpox vaccine viruses were engineered to express genes encoding the hepatitis B virus surface antigen (HBsAg), the herpes simplex virus glycoprotein D (HSV-gD) and the haemagglutinin (HA) from influenza virus. These foreign gene products when synthesized in vitro under vaccinia virus regulation were shown to be antigenic by a variety of serological tests. When these recombinant vaccinia viruses were inoculated into laboratory animals, the heterologous gene products elicited the production of specific antibodies thus demonstrating that they were immunogenic. Serum neutralizing antibodies were demonstrated to be present for both influenza and herpes simplex viruses. Additional studies in mice showed that a recombinant smallpox vaccine virus expressing a gene from herpes simplex virus effectively protected the mice when subsequently challenged with what would normally be lethal doses of infectious herpes simplex virus.     

Third-generation smallpox vaccine strain-based recombinant vaccines for viral hemorrhagic fevers

Vaccinia virus has been used as a smallpox vaccine. Now that smallpox has been eradicated, the vaccinia virus is expected to be used as a bioterrorism countermeasure and a recombinant vaccine vector for other infectious diseases, such as viral hemorrhagic fevers. Many vaccinia virus strains were used as smallpox vaccines in the smallpox eradication campaign coordinated by the World Health Organization. These strains can be classified into generations, according to the history of improving production methods and efforts to reduce the adverse reactions. Significantly, the third-generation of smallpox vaccine strains, which include modified vaccinia Ankara (MVA) and LC16m8, are currently popular as recombinant vaccine vectors due to their well-balanced safety and immunogenicity profiles. The present review firstly focuses on the characteristics of the smallpox vaccine generations. The historical background of the development of the third-generation smallpox vaccine strains is detailed, along with the history of the transition of the vaccinia virus generation used as vectors for hemorrhagic fever vaccines to the third generation. Among the vaccinia viruses, MVA is currently the most commonly used vector for developing hemorrhagic fever vaccines, including dengue fever, yellow fever, Ebola viral disease, Lassa fever, Rift Valley fever, and Crimean-Congo hemorrhagic fever. LC16m8 is a vaccine candidate for severe fever with thrombocytopenia syndrome. The current status and recent advances in the development of these hemorrhagic fever vaccines using third-generation vaccinia strains are discussed.     

The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus

The New York City Board of Health (NYCBH) vaccinia virus (VACV) vaccine strain was deleted for the immune evasion gene, E3L, and tested for its pathogenicity and ability to protect mice from heterologous challenge with ectromelia virus (ECTV). NYCBHΔE3L was found to be highly attenuated for pathogenicity in a newborn mouse model and showed a similar attenuated phenotype as the NYVAC strain of vaccinia virus. Scarification with one or two doses of the attenuated NYCBHΔE3L was able to protect mice equally as well as NYCBH from death, weight loss, and viral spread to visceral organs. A single dose of NYCBHΔE3L resulted in low poxvirus-specific antibodies, and a second dose increased levels of poxvirus-specific antibodies to a level similar to that seen in animals vaccinated with a single dose of NYCBH. However, similar neutralizing antibody titers were observed following one or two doses of NYCBHΔE3L or NYCBH. Thus, NYCBHΔE3L shows potential as a candidate for a safer human smallpox vaccine since it protects mice from challenge with a heterologous poxvirus.     

Increased attenuation but decreased immunogenicity by deletion of multiple vaccinia virus immunomodulators

Vaccinia virus (VACV)-derived vectors are popular candidates for vaccination against diseases such as HIV-1, malaria and tuberculosis. However, their genomes encode a multitude of proteins with immunomodulatory functions, several of which reduce the immunogenicity of these vectors. Hitherto only limited studies have investigated whether the removal of these immunomodulatory genes in combination can increase vaccine efficacy further. To this end we constructed viruses based on VACV strain Western Reserve (WR) lacking up to three intracellular innate immunomodulators (N1, C6 and K7). These genes were selected because the encoded proteins had known functions in innate immunity and the deletion of each gene individually had caused a decrease in virus virulence in the murine intranasal and intradermal models of infection and an increase in immunogenicity. Data presented here demonstrate that deletion of two, or three of these genes in combination attenuated the virus further in an incremental manner. However, when vaccinated mice were challenged with VACV WR the double and triple gene deletion viruses provided weaker protection against challenge. This was accompanied by inferior memory CD8⁺ T cell responses and lower neutralising antibody titres. This study indicates that, at least for the three genes studied in the context of VACV WR, the single gene deletion viruses are the best vaccine vectors, and that increased attenuation induced by deletion of additional genes decreased immunogenicity. These data highlight the fine balance and complex relationship between viral attenuation and immunogenicity. Given that the proteins encoded by the genes examined in this study are known to affect specific aspects of innate immunity, the set of viruses constructed here are interesting tools to probe the role of the innate immune response in influencing immune memory and vaccine efficacy.     

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium^® may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.     

Safety, immunogenicity and protective efficacy in mice of a new cell-cultured Lister smallpox vaccine candidate

It is now difficult to manufacture the first-generation smallpox vaccine, as the process could not comply with current safety and manufacturing regulations. In this study, a candidate non-clonal second-generation smallpox vaccine developed by Sanofi-Pasteur from the Lister strain has been assessed using a cowpox virus challenge in mice. We have observed similar safety, immunogenicity and protection (from disease and death) after a short or long interval following vaccination, as well as similar virus clearance post-challenge, with the second-generation smallpox vaccine candidate as compared to the traditional vaccine used as a benchmark.     

Smallpox--historical or real threat

Presently, there is no real possibility of natural re-emergence of smallpox virus, which was eradicated globally more then 25 years ago. During the last decade the possibility of use of smallpox virus as a biological weapon by a criminal organisation was emphasised. The re-emergence of smallpox virus would lead to unprecedented disaster. Theoretical models indicated that only extremely strict and enforced interventions could stop the spread of epidemic, but the assumptions of these models were unrealistic. Presently, there are limited stocks of the first generation smallpox vaccine left in the world. This vaccine, as well as the second-generation vaccine are associated with multiple adverse events, including fatalities and may not be accepted by society. Much safer vaccines are now being developed. Strategic plan of prophylactic vaccinations requires defining the groups to be immunised in the first place and whether immunisation should start before or after a first smallpox case would occur.     

Co-administration of tecovirimat and ACAM2000™ in non-human primates: Effect of tecovirimat treatment on ACAM2000 immunogenicity and efficacy versus lethal monkeypox virus challenge

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies’ smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000^TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.