Vaccinia Virus Infection In Mice Research Articles

Circular RNA vaccines against monkeypox virus provide potent protection against vaccinia virus infection in mice

Since the outbreak of monkeypox (mpox) in 2022, widespread concern has been placed on imposing an urgent demand for specific vaccines that offer safer and more effective protection. Using an efficient and scalable circular RNA (circRNA) platform, we constructed four circRNA vaccines that could induce robust neutralizing antibodies as well as Tcell responses by expressing different surface proteins of mpox virus (MPXV), resulting in potent protection against vaccinia virus (VACV) in mice. Strikingly, the combination of the four circular RNA vaccines demonstrated the best protection against VACV challenge among all the tested vaccines. Our study provides a favorable approach for developing MPXV-specific vaccines by using a circular mRNA platform and opens up novel avenues for future vaccine research.

Comprehensive Benchmarking of T-cell epitope prediction tools

Abstract Computational prediction tools are commonly used to select T cell epitope candidates in several immunological applications including vaccine design, cancer neoantigen identification, development of diagnostics and removal of unwanted immune responses against protein therapeutics. Most of these prediction tools are based on machine learning algorithms trained on MHC binding data or naturally processed MHC ligand data. The ability of currently available tools to predict T cell epitopes has not been comprehensively evaluated. In this study, we use a recently published dataset that systematically defined T cell epitopes recognized in vaccinia virus infected mice to evaluate the performance of all currently publicly available computational T cell epitope prediction tools. Using the same data set across all prediction methods provides an unbiased performance evaluation. The derived performance metrics allow immunologists to rationally decide on which prediction method to apply for their work at hand.

OX40 Cooperates with ICOS To Amplify Follicular Th Cell Development and Germinal Center Reactions during Infection.

Cognate interactions between T follicular helper (Tfh) cells and B cells are essential for promoting protective Ab responses. Whereas costimulatory receptors such as ICOS are accepted as being important for the induction of Tfh cell fate decision, other molecules may play key roles in amplifying or maintaining the Tfh phenotype. In this study, with vaccinia virus infection in mice, we show that OX40 was expressed on Tfh cells that accumulated at the T/B borders in the white pulp of the spleen and that OX40-dependent signals directly shaped the magnitude and quality of the their response to viral Ags. OX40 deficiency in Tfh cells profoundly impaired the acquisition of germinal center (GC) B cell phenotype, plasma cell generation, and virus-specific Ab responses. Most significantly, we found that sustained interactions between OX40 and its ligand, OX40L, beyond the time of initial encounter with dendritic cells were required for the persistence of high numbers of Tfh and GC B cells. Interestingly, OX40 was coexpressed with ICOS on Tfh cells in and around the GC, and ICOS-ICOSL interactions were similarly crucial at late times for maintenance of the Tfh and GC B cells. Thus, OX40 and ICOS act in a cooperative, nonredundant manner to maximize and prolong the Tfh response that is generated after acute virus infection.

Does CXCR3 chemokine receptor expression by CD8+ T cells affect their moving towards or only their binding to virus-infected monocytes?

This correspondence concerns a recent publication in Immunity by Hickman et al.1 who analyzed the effect of Cxcr3 knockout on migration of CD8+ T cells towards and within vaccinia virus-infected mouse ears. They found that Cxcr3 knockout had no effect on CD8+ T cell migration into the infected ears, a relatively mild effect on virus clearance, and an effect on the contact of CD8+ T cells with virus-infected cells. Curiously, despite having these basically sound and interesting data, Hickman et al. exaggerated the effect on virus clearance (“dramatically impaired virus clearance”) and focused their conclusions on assumed differences in migration towards infected cells (“CXCR3 chemokine receptor enables local CD8+ T cell migration”) rather than on better proven differences in binding to infected cells. I believe that from the data presented by Hickman et al. on the effect of Cxcr3 knockout a migration effect independent from the binding effect cannot be concluded beyond discussion. The fact that CXCR3 is a chemokine receptor, and that most researchers consequently expect a chemokine-gradient-dependent migration effect of the Cxcr3 knockout mutation, increases the risk of misleading readers when approached through the Hickman et al. narrative. The here-initiated discussion of their article may help to avoid such a misleading.

ID: 237: In virus-induced hepatitis local IFN-I responses modulate myeloid suppressor cell infiltration

Most viruses developed mechanisms that inhibit the induction or the function of type I interferon (IFN-I). Vaccinia virus (VACV), a large DNA-encoded poxvirus, encodes several IFN-I evasins, including the viral IFN-I receptor B18. Nevertheless, here we found that VACV infection still induced local IFN-I responses in secondary lymphoid organs, which are mainly sensed within the liver. VACV infected IFN-I receptor deficient (IFNAR−/−) mice developed fulminant disease, associated with high virus loads in organs such as liver and spleen, imbalanced cytokine responses within the blood, and eventually succumbed to infection. Elevated ALT/AST levels in the serum of VACV infected IFNAR−/− mice supported the hypothesis that IFNAR triggering of the liver played a major role in the antiviral response. Histological analysis of livers of VACV infected IFNAR−/− mice revealed a severe acute hepatitis, massive infiltration of immune cells, and acute tissue apoptosis in the absence of IFNAR-signaling. Interestingly, VACV infected mice with a hepatocyte selective IFNAR ablation (Alb-CreIFNARflox/flox) showed moderate hepatitis as observed in wild-type mice, whereas mice with a cell type-specific IFNAR deletion only in myeloid cells (LysM-CreIFNARflox/flox) showed enhanced hepatitis. Thus, local IFN-I responses sensed by myeloid cells prevented immunopathology within the liver, presumably due to the induction of myeloid derived suppressor cells (MDSC). Collectively these results indicate that although the induction of serum IFN-I is sequestered, VACV infection induces local IFN-I responses that play a central role in modulating immune responses and thus in reducing immunopathology and hepatitis.

Protection against lethal vaccinia virus infection in mice using an siRNA targeting the A5R gene.

Although the World Health Organization has declared the eradication of smallpox in 1980, the fear of its potential use in bioterrorism has become a reality. Since the effectiveness of current vaccines and antiviral drugs is limited, development of new therapeutic strategies is needed. In this study, we investigated small interfering RNA (siRNA) as a therapeutic approach for preventing and treating smallpox infection. Eight siRNA sequences were designed and evaluated for antiviral activity against vaccinia virus (VACV) in vitro and in vivo. Of eight siRNAs, A5R1 siRNA targeted the A5R gene and reduced VACV replication in cell culture by up to 85% at 100 nM concentration without inducing cytotoxicity. A prolonged prophylactic as well as therapeutic effect of siRNA was observed. In addition, real-time PCR analysis showed that A5R1 siRNA can especially reduce the target mRNA. Finally, intraperitoneal delivery of A5R1 siRNA in Balb/c mice significantly protected these animals from lethal challenge with VACV. This study suggests the potential of A5R1 siRNA as a therapeutic antiviral agent against smallpox.

CD8 T cells use IFN-γ to protect against the lethal effects of a respiratory poxvirus infection.

CD8 T cells are a key component of immunity to many viral infections. They achieve this through using an array of effector mechanisms, but precisely which component/s are required for protection against a respiratory orthopox virus infection remains unclear. Using a model of respiratory vaccinia virus infection in mice, we could specifically determine the relative contribution of perforin, TRAIL, and IFN-γ-mediated pathways in protection against virus induced morbidity and mortality. Unexpectedly, we observed that protection against death was mediated by IFN-γ without any involvement of the perforin or TRAIL-dependent pathways. IFN-γ mRNA and protein levels in the lung peaked between days 3 and 6 postinfection. This enhanced response coincided with the emergence of virus-specific CD8 T cells in the lung and the cessation of weight loss. Transfer experiments indicated that CD8 T cell-autonomous expression of IFN-γ restricts virus-induced lung pathology and dissemination to visceral tissues and is necessary for clearance of virus. Most significantly, we show that CD8 T cell-derived IFN-γ is sufficient to protect mice in the absence of CD4 and B-lymphocytes. Thus, our findings reveal a previously unappreciated mechanism by which effector CD8 T cells afford protection against a highly virulent respiratory orthopox virus infection.

IL-12 is required for mTOR regulation of memory CTLs during virus infection (P6232)

Abstract Inflammatory cytokines are critical in the induction of memory CTLs, and inhibition of mTOR by rapamycin can enhance memory CTL generation. However, it is unknown if important inflammatory cytokines such as IL-12 are involved in the regulation of memory CTLs by mTOR during infection. We show here that inhibition of mTOR by rapamycin represses CTLs expansion and enhances memory CTL generation during vaccinia virus infection in mice. In addition, rapamycin promotes central memory phenotype in CTLs. However, the absence of IL-12 signal in CTLs diminishes rapamycin regulation on both CTL expansion and subsequent memory CTL formation, despite that rapamycin substantially increases memory CTLs when IL-12 signal is missing. Moreover, rapamycin-regulated memory CTLs in the absence of IL-12 are impaired in secondary expansion upon pathogen challenge, and subsequently form abolished secondary memory. In conclusion, IL-12 contributes to the strength of rapamycin regulation on primary memory CTLs, and lack of IL-12 signal leads to impaired secondary expansion of memory CTLs. Thus, the IL-12 signal is required for mTOR regulation on functional memory CTLs.

The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice

In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8(+) T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8(+) T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination.

Increased Protection from Vaccinia Virus Infection in Mice Genetically Prone to Lymphoproliferative Disorders

Mutations in the genes that encode Fas or Fas ligand (FasL) can result in poor restraints on lymphocyte activation and in increased susceptibility to autoimmune disorders. Because these mutations portend a continuously activated immune state, we hypothesized that they might in some cases confer resistance to infection. To examine this possibility, the immune response to, morbidity caused by, and clearance of vaccinia virus (VACV) Western Reserve was examined in 5- to 7-week-old Fas mutant (lpr) mice, before an overt lymphoproliferative disorder was observable. On day 6 after VACV infection, C57BL/6-lpr (B6-lpr) mice had decreased morbidity, decreased viral titers, and an increased percentage and number of CD4(+) and CD8(+) T cells. As early as day 2 after infection, B6-lpr mice had decreased liver and spleen viral titers and increased numbers of and increased gamma interferon (IFN-γ) production by several different effector cell populations. Depletion of individual effector cell subsets did not inhibit the resistance of B6-lpr mice. Uninfected B6-lpr mice also had increased numbers of NK cells, γδ(+) T cells, and CD44(+) CD4(+) and CD44(+) CD8(+) T cells compared to uninfected B6 mice. Antibody to IFN-γ resulted in increased virus load in both B6 and B6-lpr mice and eliminated the differences in viral titers between them. These results suggest that IFN-γ produced by multiple activated leukocyte populations in Fas-deficient hosts enhances resistance to some viral infections.

Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

BackgroundPreviously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV).ResultsHere, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality.ConclusionsA combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs.

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.

Adverse Events Post Smallpox-Vaccination: Insights from Tail Scarification Infection in Mice with Vaccinia virus

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1 −/−) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1 −/− with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1 −/−, and passive transfer of WT T cells to Rag1 −/− animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.

Cannabinoids lead to enhanced virulence of the smallpox vaccine (vaccinia) virus

Indian hemp is used since thousands of years as herbal drug. We found that a single dose of cannabis resin was equally active as Δ9-tetrahydrocannabinol (THC) enhancing severity and duration of symptoms in vaccinia virus infected mice. Cowpox virus did not cause symptomatic disease, but some reduction of specific antibody production was observed in drug treated animals. In vitro cannabis was superior to THC alone at inhibiting mitogen stimulated proliferation of human and mouse spleen cells and peripheral blood mononuclear cells. Also resin sub-fractions other than THC, cannabidiol and cannabinol, recovered also from cigarette smoke, were found inhibitory, suggesting additional involvement of constituents other than psychoactive THC. The immunoregulatory effects must be differentiated from apoptotic effects on spleen cells and lymphocytic mouse cell lines, which were observed with resin and THC but not with cannabidiol or cannabinol. A significant contribution of cytotoxic effects seems unlikely as drug treated lymphocytes were still capable of producing cytokines after T-cell receptor-specific stimulation. Considering a recent case of unusually severe cowpox virus infection in a young drug taker these data confirm a risk of “soft drugs” for acquiring poxvirus infection or enhancing side effects of the smallpox vaccine and perhaps also other live vaccines.

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.

Lack of Efficacy of Aurintricarboxylic Acid and Ethacrynic Acid against Vaccinia Virus Respiratory Infections in Mice

Aurintricarboxylic acid (ATA) and ethacrynic acid (ECA) have been reported to exhibit antiviral activity against vaccinia virus infections in cell culture by inhibiting early and late gene transcription, respectively. The purpose of this work was to determine if these inhibitors would effectively treat vaccinia virus infections in mice, which has not previously been studied. ECA was investigated by cell culture plaque reduction assay for the inhibition of cowpox and vaccinia virus infections to clarify issues regarding its potency and selectivity. Mice infected intranasally with vaccinia virus were treated by intraperitoneal route twice daily for 5 days with ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) or once daily for 2 days with cidofovir (100 mg/kg/day). ECA caused 50% inhibition of virus plaque formation at 20-79 muM in four cultured cell lines, with 50% cytotoxicity at 84-173 muM, giving low (1.3-4.2) selectivity index values. Preliminary toxicity tests in uninfected mice indicated that ATA and ECA were both overtly toxic at 100 mg/kg/day. No protection from mortality was afforded by treatment of vaccinia virus infections with ATA or ECA, but 100% survival was achieved in the cidofovir group. ATA- and ECA-treated mice died significantly sooner than placebo-treated animals, indicating that these compounds exacerbated the infection. Both ATA and ECA lack antiviral potency and selectivity in cell culture. The compounds were ineffective in treating mice at intraperitoneal doses of <or=30 mg/kg/day. These compounds do not appear to have potential for the treatment of poxvirus infections in vivo.

Application of Bioluminescence Imaging to the Prediction of Lethality in Vaccinia Virus-Infected Mice

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC = 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.

Effect of Oral Treatment with Hexadecyloxypropyl-[( S )-9-(3-Hydroxy-2- Phosphonylmethoxypropyl)Adenine] [( S )-HPMPA] or Octadecyloxyethyl-( S )-HPMPA on Cowpox or Vaccinia Virus Infections in Mice

We have previously reported that (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, or (S)-HPMPA, is active in vitro against cowpox virus (CV) and vaccinia virus (VV) but is not active orally in animals. However, the ether lipid esters of (S)-HPMPA, hexadecyloxypropyl-[(S)-HPMPA] [HDP-(S)-HPMPA] and octadecyloxyethyl-[(S)-HPMPA] [ODE-(S)-HPMPA], had significantly enhanced activity in vitro and are orally bioavailable in mice. In the current study, HDP-(S)-HPMPA and ODE-(S)-HPMPA were prepared in water and administered once daily by oral gavage to mice at doses of 30, 10, and 3 mg/kg of body weight for 5 days beginning 24, 48, or 72 h after inoculation with CV or VV. Oral HDP-(S)-HPMPA and ODE-(S)-HPMPA were both highly effective (P < 0.001) at preventing mortality due to CV at 30 mg/kg, even when treatments were delayed until up to 72 h postinfection. ODE-(S)-HPMPA or HDP-(S)-HPMPA were also highly effective (P < 0.001) at preventing mortality in mice infected with VV at 30 mg/kg when treatments were delayed until to 48 or 72 h postinfection, respectively. Protection against both viruses was associated with a significant reduction of virus replication in the liver, spleen, and kidney but not in the lung. These data indicate that HDP-(S)-HPMPA and ODE-(S)-HPMPA are active when given orally against lethal CV and VV infections in mice, and further evaluation is warranted to provide additional information on the potential of these orally active compounds for treatment of human orthopoxvirus infection.

Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions.

The WHO smallpox eradication program was concluded 21 years ago and the non-vaccinated population is now at risk of poxvirus infections, either by contact with monkeypox or through bioterrorism. Since drugs specific against poxvirus infections are limited, neutralizing monoclonal antibodies (mAbs) that are effective in vivo may be an important tool in controlling poxvirus infections. To this end, we studied the efficacy of the mAb C3, reactive against the trimeric 14-kDa protein of vaccinia virus (VV) localized in the membrane of the intracellular form of mature virus, for its ability to neutralize VV infection in mice. The results show that prophylactic as well as therapeutic administration of mAb C3 can be an effective means of control of VV replication within the host. The interval of antibody efficacy following a single administration, before and after VV inoculation, has been defined. This study reinforces the notion that neutralizing mAbs should be developed to control health-related human infections by poxviruses.