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Modified Vaccinia Ankara Bavarian Nordic (MVA-BN) as a smallpox and mpox vaccine has been approved in its liquid-frozen (LF) formulation in the US, Canada, and EU. A freeze-dried (FD) formulation may offer additional benefits, such as a longer shelf life and reduced dependence on cold chain storage and transport. In a phase 2 clinical trial, 651 vaccinia-naïve participants were vaccinated with two doses of MVA-BN LF or FD, 4 weeks apart. The objectives were to compare MVA-BN FD with LF in terms of vaccine-induced immune responses, safety, and reactogenicity. Non-inferiority of the immune response was assessed by the 95% CI of the geometric mean ratios. Both formulations induced robust vaccinia-specific humoral and cellular immune responses. At peak humoral responses (Week 6), geometric means of total antibody titers were 1096 (95% CI 1013, 1186) from the FD group and 877 (95% CI 804, 956) from the LF group, achieving the primary endpoint of non-inferiority of MVA-BN FD compared to MVA-BN LF. At peak cellular responses (Week 2), geometric means of T cell spot forming units were 449 (95% CI 341, 590) from the FD group and 316 (95% CI 234, 427) from the LF group. Both formulations of MVA-BN were well tolerated, with similar unsolicited AEs and solicited systemic reactions in both groups but slightly more local reactions in the FD group. No vaccine-related serious adverse events (SAEs) or vaccine-related AE of special interest were reported. The FD formulation of MVA-BN was shown to be equivalent to MVA-BN LF.

Recombinant chimeric horsepox virus (TNX-801) is a preclinical vaccine in development against mpox and smallpox. In this report, we investigated the potential phenotypic differences in in vitro and in vivo models between TNX-801 and older vaccinia virus (VACV)-based vaccine strains (VACV-Lis and VACV-NYCBH) used in the eradication of smallpox as well as VACV-WR, VACV-IHD, and MVA. TNX-801 displayed a small plaque phenotype (~1-2 mm) in BSC-40 and Vero-E6 cells. Multi-step replication kinetics in immortalized nonhuman primate cell lines, and human primary cells from dermal and respiratory tracts yielded >10- to 100-fold lower infectious titers than the VACV strains. In addition, the infectious particle-to-genome copy ratio data suggests that TNX-801 genome packaging is ~10- to 100-fold less efficient than the VACV strains and the potential mechanism of TNX-801 attenuation is at the packaging/egress stage. Lastly, the susceptibility to VACV and TNX-801 infection of three new immunocompromised murine models (C56BL/6 Ifnar -/- , C56BL/6 Ifngr -/- , and C56BL/6 Ifnar -/- / Ifngr -/- ) was investigated. VACV strains were able to produce severe disease including decrease in body weight and temperature, as well as lethality in murine models via the intraperitoneal or intranasal routes. In contrast to VACV strains, TNX-801 was unable to produce any disease in murine models. These data demonstrate that TNX-801 is >10- to 1,000-fold more attenuated compared to older VACV-based smallpox vaccine strains in human primary cell lines and immunocompromised mice., Importance: Variola and monkeypox viruses are medically important pathogens that can cause fatal human disease. The two FDA-approved vaccines, ACAM-2000 and JYNNEOS, have important advantages and disadvantages. ACAM-2000 offers durable immunity; however, it has high adverse event rates. In contrast, JYNNEOS has a safer profile but requires two doses 4-weeks apart to achieve comparable immunity. Consequently, there is a need for vaccines offering durable immunity via single immunization with minimal adverse events. TNX-801 is a preclinical stage vaccine that can stimulate potent immunity via a single dose and provides protection against lethal mpox disease in the nonhuman primate model. Here, we show that TNX-801 is >10- to 1,000-fold attenuated in in vitro and in vivo models including human primary cells and immunocompromised murine models than vaccine strains utilized in smallpox eradication. The natural attenuation of TNX-801 and its ability to induce protective immunity via a single vaccination are promising and warrants further development., Competing Interests: Seth Lederman is a co-inventor of the TNX-801 vaccine described in this study.

Background: Mpox is a disease endemic to Central and West Africa. It caused outbreaks in non-endemic countries, mainly in 2022. The endemic Democratic Republic of Congo is currently experiencing its largest outbreak yet. The vaccine Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) is approved for active immunization against mpox and smallpox. Since the outbreak in 2022, real-world studies have assessed MVA-BN's vaccine effectiveness (VE) against mpox, and this systematic literature review aims to summarize the most current evidence., Methods: Medline (via PubMed), Embase, and LILACS were searched, as well as grey literature sources and publications' bibliographies to identify observational studies published between 1/Jan/2022 and 28/Feb/2024 that estimate the VE of MVA-BN against mpox or provide risk measures that allow calculation of these VE estimates. Data were presented descriptively in tables and text; the methodological quality of included records was assessed using NHLBI/NIH quality assessment tools., Results: The literature search identified 16 records that fit the inclusion criteria. The studies took place in high-income countries and were heterogeneous in design, setting, and definition of at-risk populations. MVA-BN VE estimates against mpox infection were assessed. Where the study population was exclusively or primarily those receiving pre-exposure prophylactic vaccination, the adjusted VE estimates ranged from 35 % to 86 % (n = 8 studies) for one dose and from 66 % to 90 % (n = 5) for two doses. Where only post-exposure prophylactic vaccination was assessed, adjusted VE estimates were reported for one dose only at 78 % and 89 % (n = 2). Additionally, MVA-BN reduced the risk of mpox-related hospitalization in one study and the severity of mpox clinical manifestations in two studies., Conclusions: Despite heterogeneity in study design, setting, and at-risk populations, the reported VE estimates against mpox infection demonstrated the effectiveness of one or two doses of MVA-BN in the context of an outbreak across multiple countries., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Suzanne Scheele and Florian Lienert report financial support was provided by Bavarian Nordic. Suzanne Scheele and Florian Lienert report a relationship with Bavarian Nordic that includes: employment. The study-related activities of P95 employees Lauren M.K. Mason, Estefania Betancur, Margarita Riera-Montes were funded by Bavarian Nordic., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A large outbreak of monkeypox occurred in 2022, and most people lack immunity to orthopoxvirus. Smallpox vaccination is essential for preventing further smallpox outbreaks. This study evaluated the effectiveness, protection, safety, and cross-immunogenicity of smallpox vaccine in preventing monkeypox infection. PubMed, Embase, Scopus, and Web of Science were searched from database inception to 10 March 2024. We included studies involving "monkeypox virus" and "vaccinations", and excluded reviews, animal studies, and articles with missing or duplicate data. A total of 37 studies with 57,693 participants were included in the final analysis. The effectiveness data showed that monkeypox infection rates were lower in the smallpox-vaccinated group than in the unvaccinated group (risk ratio [RR]: 0.46; 95% confidence interval [CI]: 0.31-0.68). The protection data showed that smallpox vaccination effectively reduced the risk of severe monkeypox infection (RR: 0.61; 95% CI: 0.42-0.87). Third-generation vaccines showed greater efficacy (RR: 0.36, 95% CI: 0.22-0.56) than first-generation vaccines. The number of doses of smallpox vaccine has no significant effect on monkeypox. Safety data showed that adverse reactions after smallpox vaccination were mainly mild and included local erythema, swelling, induration, itching, and pain. Meanwhile, we found that smallpox vaccination could induce the production of neutralizing antibodies against monkeypox. Our findings offer compelling evidence supporting the clinical application of the smallpox vaccine for preventing monkeypox and advocate that high-risk groups should be prioritized for receiving one dose of the smallpox vaccine if the vaccine stockpile is low.

Men who have sex with men and people living with HIV are disproportionately affected in the 2022 multi-country monkeypox epidemic. The smallpox vaccine can induce cross-reactive antibodies against the monkeypox virus (MPXV) and reduce the risk of infection. Data on antibodies against MPXV induced by historic smallpox vaccination in people with HIV are scarce. In this observational study, plasma samples were collected from people living with and without HIV in Shenzhen, China. We measured antibodies binding to two representative proteins of vaccinia virus (VACV; A27L and A33R) and homologous proteins of MPXV (A29L and A35R) using an enzyme-linked immunosorbent assay. We compared the levels of these antibodies between people living with and without HIV. Stratified analyses were performed based on the year of birth of 1981 when the smallpox vaccination was stopped in China. Plasma samples from 677 people living with HIV and 746 people without HIV were tested. A consistent pattern was identified among the four antibodies, regardless of HIV status. VACV antigen-reactive and MPXV antigen-reactive antibodies induced by historic smallpox vaccination were detectable in the people born before 1981, and antibody levels reached a nadir during or after 1981. The levels of smallpox vaccine-induced antibodies were comparable between people living with HIV and those without HIV. Our findings suggest that the antibody levels against MPXV decreased in both people living with and without HIV due to the cessation of smallpox vaccination.

Mpox is currently a global health emergency. This review (Part II) aims to provide insights into Mpox vaccines and their advancements, offering easily digestible information for healthcare workers and researchers. Current Mpox vaccines are all live-attenuated, previously approved for smallpox, and are classified into non-replicating (Modified Vaccinia Ankara-Bavarian Nordic or MVA-BN) and replicating vaccines (Lister clone16m8 KM Biologic or LC16m8KMB and Acambis2000 or ACAM2000). Replicating vaccines offer long-lasting immunity but are contraindicated for immunocompromised individuals and those with extensive dermatitis. Replicating vaccines are administered as a single dose via epicutaneous scarification, while the non-replicating vaccine is given as two subcutaneous doses. Regulatory approvals in various countries are based on animal challenge studies, with limited effectiveness data available. Only LC16m8 is approved for children in Japan, while the others are approved for individuals aged 18 and older. Clinical trials are currently investigating the efficacy and safety of MVA-BN, particularly in children and for post-exposure prophylaxis (PEP). Novel Mpox vaccines that provide cross-protection against orthopoxviruses are needed, with DNA, subunit, and mRNA platforms under development. MPXV-neutralizing antibody-inducing target antigens for vaccine development include the outer envelope antigens of extracellular enveloped virus (EEV): A35R and B6R, and the inner membrane antigens of intracellular mature virus (IMV): M1R, A29L, H3L, and E8L. Two mRNA vaccines are currently in early clinical stages. Importantly, the COVID-19 pandemic underscored the importance of addressing vaccine disparities and improving global access. Transformative approaches are being explored to overcome this challenge and to enhance access in low- and middle-income countries.

Skin scar formation following Bacille Calmette-Guérin (BCG) or smallpox (Vaccinia) vaccination is an established marker of successful vaccination and 'vaccine take'. Potent pathogen-specific (tuberculosis; smallpox) and pathogen-agnostic (protection from diseases unrelated to the intentionally targeted pathogen) effects of BCG and smallpox vaccines hold significant translational potential. Yet despite their use for centuries, how scar formation occurs and how local skin-based events relate to systemic effects that allow these two vaccines to deliver powerful health promoting effects has not yet been determined. We review here what is known about the events occurring in the skin and place this knowledge in the context of the overall impact of these two vaccines on human health with a particular focus on maternal-child health., (© 2024. The Author(s).)

Potential threat of smallpox bioterrorism and concerns related to the adverse effects of currently licensed live-virus vaccines suggest the need to develop novel vaccines with better efficacy against smallpox. Use of DNA vaccines containing specific antigen-encoding plasmids prevents the risks associated with live-virus vaccines, offering a promising alternative to conventional smallpox vaccines. In this study, we investigated the efficiency of toll-like receptor (TLR) ligands in enhancing the immunogenicity of smallpox DNA vaccines. BALB/c mice were immunized with a DNA vaccine encoding the vaccinia virus L1R protein, along with the cytosine-phosphate-guanine (CpG) motif as a vaccine adjuvant, and their immune response was analyzed. Administration of B-type CpG oligodeoxynucleotides (ODNs) as TLR9 ligands 24 h after DNA vaccination enhanced the Th2-biased L1R-specific antibody immunity in mice. Moreover, B-type CpG ODNs improved the protective effects of the DNA vaccine against the lethal Orthopoxvirus challenge. Therefore, use of L1R DNA vaccines with CpG ODNs as adjuvants is a promising approach to achieve effective immunogenicity against smallpox infection., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body., Importance: In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent., Competing Interests: The authors declare no conflict of interest.

Since May 2022, several countries outside of Africa experienced multiple clusters of monkeypox virus (MPXV)-associated disease. In the present study, anti-MPXV and anti-vaccinia virus (VACV) neutralizing antibody responses were evaluated in two cohorts of subjects from the general Italian population (one half born before the WHO-recommended end of smallpox vaccination in 1980, the other half born after). Higher titers (either against MPXV or VACV) were observed in the cohort of individuals born before the interruption of VACV vaccination. An association between VACV and MPXV antibody levels was observed, suggesting that the smallpox vaccination may confer some degree of cross-protection against MPXV infection. Results from this study highlight low levels of immunity toward the assessed Orthopoxviruses, especially in young adults, advocating the introduction of a VACV- or MPXV-specific vaccine in case of resurgence of monkeypox disease outbreaks., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: After the eradication of smallpox, there have been no specific public health measures for any Orthopoxviruses (OPXVs). Therefore, it is necessary to countermeasure OPXV infections after Mpox (formerly monkeypox) occurrences, such as the latest global outbreak in 2022-2023. This study aimed to provide crucial insights for the development of effective public health policy making against mpox in populations residing in regions where the virus is not prevalent., Methods: This study used enzyme-linked immunosorbent assays (ELISA) to examine smallpox and mpox antibodies in Koreans with three different age groups. We analyzed 56 sera obtained from a tertiary care hospital in South Korea between September 2022 and April 2023. Plasma levels of antibodies against the viral proteins of smallpox (variola cytokine response-modifying protein B) and MPXV (A29) were measured using enzyme-linked immunosorbent assays., Results: Plasma samples from participants in their early 40 s and older exhibited higher reactivity to viral antigens than those from younger participants. Furthermore, there was a strong positive correlation in antibody positivity for the two different viruses across the sera., Conclusions: The presence of low antibody levels in participants ˂40 years may hinder their ability to defend against OPXV. Therefore, it is imperative to implement effective public health measures to mitigate the transmission of OPXV within the community. These findings serve as fundamental information for devising strategies to combat mpox efficiently, particularly in regions where the virus is not prevalent., Competing Interests: Declaration of Competing Interest The authors have declared no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Increased human-to-human transmission of monkeypox virus (MPXV) is cause for concern, and antibodies directed against vaccinia virus (VACV) are known to confer cross-protection against Mpox. We used 430 serum samples derived from the Scottish patient population to investigate antibody-mediated cross-neutralization against MPXV. By combining electrochemiluminescence immunoassays with live-virus neutralization assays, we show that people born when smallpox vaccination was routinely offered in the United Kingdom have increased levels of antibodies that cross-neutralize MPXV. Our results suggest that age is a risk factor of Mpox infection, and people born after 1971 are at higher risk of infection upon exposure.

Aims: To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory., Methods: Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry., Results: None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals., Conclusions: ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.

The smallpox infection with the variola virus was one of the most fatal disorders until a global eradication was initiated in the twentieth century. The last cases were reported in Somalia 1977 and as a laboratory infection in the UK 1978; in 1980, the World Health Organization (WHO) declared smallpox for extinct. The smallpox virus with its very high transmissibility and mortality is still a major biothreat, because the vaccination against smallpox was stopped globally in the 1980s. For this reason, new antivirals (cidofovir, brincidofovir, and tecovirimat) and new vaccines (ACAM2000, LC16m8 and Modified Vaccine Ankara MVA) were developed. For passive immunization, vaccinia immune globulin intravenous (VIGIV) is available. Due to the relationships between orthopox viruses such as vaccinia, variola, mpox (monkeypox), cowpox, and horsepox, the vaccines (LC16m8 and MVA) and antivirals (brincidofovir and tecovirimat) could also be used in the mpox outbreak with positive preliminary data. As mutations can result in drug resistance against cidofovir or tecovirimat, there is need for further research. Further antivirals (NIOCH-14 and ST-357) and vaccines (VACΔ6 and TNX-801) are being developed in Russia and the USA. In conclusion, further research for treatment and prevention of orthopox infections is needed and is already in progress. After a brief introduction, this chapter presents the smallpox and mpox disease and thereafter full overviews on antiviral treatment and vaccination including the passive immunization with vaccinia immunoglobulins., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Smallpox was a significant cause of mortality for over three thousand years, amounting to 10% of deaths yearly. Edward Jenner discovered smallpox vaccination in 1796, which rapidly became a smallpox infection preventive practice throughout the world and eradicated smallpox infection by 1980. After smallpox eradication, monkeypox vaccines have been used primarily in research and in outbreaks in Africa, where the disease is endemic. In the present, the vaccines are being used for people who work with animals or in high-risk areas, as well as for healthcare workers treating patients with monkeypox. Among all orthopoxviruses (OPXV), monkeypox viral (MPXV) infection occurs mainly in cynomolgus monkeys, natural reservoirs, and occasionally causes severe multi-organ infection in humans, who were the incidental hosts. The first case of the present epidemic of MXPV was identified on May 7, 2022, and rapidly increased the number of cases. In this regard, the WHO declared the outbreak, an international public health emergency on July 23, 2022. The first monkeypox vaccine was developed in the 1960s by the US Army and was based on the vaccinia virus, which is also used in smallpox vaccines. In recent years, newer monkeypox vaccines have been developed based on other viruses such as Modified Vaccinia Ankara (MVA). These newer vaccines are safer and can provide longer-lasting immunity with fewer side effects. For the future, there is ongoing research to improve the current vaccines and to develop new ones. One notable advance has been the development of a recombinant vaccine that uses a genetically modified vaccinia virus to express monkeypox antigens. This vaccine has shown promising results in pre-clinical trials and is currently undergoing further testing in clinical trials. Another recent development has been the use of a DNA vaccine, which delivers genetic material encoding monkeypox antigens directly into cells. This type of vaccine has shown effectiveness in animal studies and is also undergoing clinical testing in humans. Overall, these recent advances in monkeypox vaccine development hold promise for protecting individuals against this potentially serious disease., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background Aims: E3L is an immediate-early protein of vaccinia virus (VV) that is detected within 0.5 h of infection, potentially before the many immune evasion genes of vaccinia can exert their protective effects. E3L is highly conserved among orthopoxviruses and hence could provide important protective T-cell epitopes that should be retained in any subunit or attenuated vaccine. We have therefore evaluated the immunogenicity of E3L in healthy VV-vaccinated donors., Methods: Peripheral blood mononuclear cells from healthy volunteers (n = 13) who had previously received a smallpox vaccine (Dryvax) were activated and expanded using overlapping E3L peptides and their function, specificity and antiviral activity was analyzed. E3L-specific T cells were expanded from 7 of 12 (58.3%) vaccinated healthy donors. Twenty-five percent of these produced CD8+ T-cell responses and 87.5% produced CD4+ T cells. We identified epitopes restricted by HLA-B35 and HLA-DR15., Results: E3L-specific T cells killed peptide-loaded target cells as well as vaccinia-infected cells, but only CD8+ T cells could prevent the spread of infectious virus in virus inhibition assays. The epitopes recognized by E3L-specific T cells were shared with monkeypox, and although there was a single amino acid change in the variola epitope homolog, it was recognized by vaccinia-specific T-cells., Conclusions: It might be important to include E3L in any deletion mutant or subunit vaccine and E3L could provide a useful antigen to monitor protective immunity in humans., Competing Interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (38; Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The re-emergence of smallpox is an increasing and legitimate concern due to advances in synthetic biology. Vaccination programs against smallpox using the vaccinia virus vaccine ceased with the eradication of smallpox and, unlike many other countries, Australia did not use mass vaccinations. However, vaccinated migrants contribute to population immunity. Testing for vaccinia antibodies is not routinely performed in Australia, and few opportunities exist to estimate the level of residual population immunity against smallpox. Serological data on population immunity in Australia could inform management plans against a smallpox outbreak. Vaccinia antibodies were measured in 2003 in regular plasmapheresis donors at the Australian Red Cross Blood Service from New South Wales (NSW). The data were analysed to estimate the proportion of Australians in NSW with detectable serological immunity to vaccinia. The primary object of this study was to measure neutralising antibody titres against vaccinia virus. Titre levels in donor samples were determined by plaque reduction assay. To estimate current levels of immunity to smallpox infection, the decline in geometric mean titres (GMT) over time was projected using two values for the antibody levels estimated on the basis of different times since vaccination. The results of this study suggest that there is minimal residual immunity to the vaccinia virus in the Australian population. Although humoral immunity is protective against orthopoxvirus infections, cell-mediated immunity and immunological memory likely also play roles, which are not quantified by antibody levels. These data provide an immunological snapshot of the NSW population, which could inform emergency preparedness planning and outbreak control, especially concerning the stockpiling of vaccinia vaccine.

The ancient technique of variolation (inoculation of the smallpox) which was introduced in the United States in 1721 was replaced by vaccination (inoculation of the cowpox) soon after the procedure was published by Edward Jenner in 1798. Benjamin Waterhouse is recognized as the introducer of smallpox vaccination in the United States having conducted the first vaccination in Boston on 8 July 1800, although other American physicians also played an important role in extending vaccination in the East Coast of the United States. A different route of introduction brought the smallpox vaccine from Mexico to New Mexico (March 1805) and Texas (April 1806) which at that time where part of the Viceroyalty of New Spain. The vaccine was brought to California in 1817 by Russian merchants who obtained it in Peru, where the vaccine had arrived in 1806 with the Spanish Philanthropic Expedition of the Vaccine. It took almost 150 years of vaccination efforts before the last natural outbreak of smallpox occurred in the United States in 1949., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Numerous animal models of systemic orthopoxvirus disease have been developed to evaluate therapeutics against variola virus (VARV), the causative agent of smallpox. These animal models do not resemble the disease presentation in human smallpox and most used surrogate Orthopoxviruses. A rodent model using VARV has a multitude of advantages, and previous investigations identified the CAST/EiJ mouse as highly susceptible to monkeypox virus infection, making it of interest to determine if these rodents are also susceptible to VARV infection. In this study, we inoculated CAST/EiJ mice with a range of VARV doses (10 2 -10 6 plaque forming units). Some animals had detectable viable VARV from the oropharynx between days 3 and 12 post inoculation. Despite evidence of disease, the CAST/EiJ mouse does not provide a model for clinical smallpox due to mild signs of morbidity and limited skin lesions. However, in contrast to previous rodent models using VARV challenge (i.e. prairie dogs and SCID mice), a robust immune response was observed in the CAST/EiJ mice (measured by Immunoglobulin G enzyme-linked immunosorbent assay). This is an advantage of this model for the study of VARV and presents a unique potential for the study of the immunomodulatory pathways following VARV infection., (Published by Elsevier B.V.)

Background: Many countries have stockpiled vaccines because of concerns about the reemergence of smallpox. Traditional smallpox vaccines are based on replicating vaccinia viruses; these vaccines have considerable side effects., Methods: To evaluate the efficacy of modified vaccinia Ankara (MVA) as a potential smallpox vaccine, we randomly assigned 440 participants to receive two doses of MVA followed by one dose of the established replicating-vaccinia vaccine ACAM2000 (the MVA group) or to receive one dose of ACAM2000 (the ACAM2000-only group). The two primary end points were noninferiority of the MVA vaccine to ACAM2000 with respect to the peak serum neutralizing antibody titers and attenuation of the ACAM2000-associated major cutaneous reaction by previous MVA vaccination, measured according to the maximum lesion area and the derived area attenuation ratio., Results: A total of 220 and 213 participants were randomly assigned and vaccinated in the MVA group and ACAM2000-only group, respectively, and 208 participants received two MVA vaccinations. At peak visits, MVA vaccination induced a geometric mean titer of neutralizing antibodies of 153.5 at week 6, as compared with 79.3 at week 4 with ACAM2000 (a ratio of 1.94 [95% confidence interval {CI}, 1.56 to 2.40]). At day 14, the geometric mean titer of neutralizing antibodies induced by a single MVA vaccination (16.2) was equal to that induced by ACAM2000 (16.2), and the percentages of participants with seroconversion were similar (90.8% and 91.8%, respectively). The median lesion areas of the major cutaneous reaction were 0 mm 2 in the MVA group and 76.0 mm 2 in the ACAM2000-only group, resulting in an area attenuation ratio of 97.9% (95% CI, 96.6 to 98.3). There were fewer adverse events or adverse events of grade 3 or higher after both MVA vaccination periods in the MVA group than in the ACAM2000-only group (17 vs. 64 participants with adverse events of grade 3 or higher, P<0.001)., Conclusions: No safety concerns associated with the MVA vaccine were identified. Immune responses and attenuation of the major cutaneous reaction suggest that this MVA vaccine protected against variola infection. (Funded by the Office of the Assistant Secretary for Preparedness and Response Biomedical Advanced Research and Development Authority of the Department of Health and Human Services and Bavarian Nordic; ClinicalTrials.gov number, NCT01913353.)., (Copyright © 2019 Massachusetts Medical Society.)

Concerns about bioterrorism and outbreaks of zoonotic orthopoxvirus require safe and efficacious smallpox vaccines. We previously reported the clinical efficacy and safety profiles of LC16m8, a live, attenuated, cell culture-derived, smallpox vaccine, examined in over 3000 healthy Japanese adults with various vaccination histories. In this study, serum of approximately 200 subjects pre and post LC16m8 vaccination were subjected to a vaccinia virus-specific protein array to evaluate the proteome-wide immunogenicity. The relationships between antigen-specific antibodies and plaque reduction neutralization titers were analyzed. LC16m8 induced antibodies to multiple vaccinia antigens in primary-vaccinated individuals and yielded effective booster responses in previously vaccinated individuals, demonstrating similar antibody profiles to those reported for other vaccinia virus strains. Several immunodominant antigens were indicated to be important for neutralization of the intracellular mature virion. The similarity of antibody profiles between LC16m8 and other smallpox vaccine strains supports the immunogenicity and protective efficacy of LC16m8., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Based on archival materials, collected literature and archival articles analysing the clinical course of the disease, the article presents the medical and social course of one of the recent epidemics of smallpox in Europe, which took place in Wrocław in 1963. During the epidemic, 99 people fell ill and seven of them died. The authors describe how a mass vaccination campaign was organised in the city and the entire surrounding region. This historical study shows not only the course of the epidemic itself, but also the ways to prevent and deal with infectious diseases and the organisation of vaccinations in communist Poland. The authors also discuss the issue of the relationship between the vaccination period and the course of smallpox in patients and show the scale of post-vaccination complications in the situation of mass vaccination against smallpox. Although the article refers to historical events, it draws attention to the topicality of challenges posed by the variola virus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Edward Jenner's discovery of the smallpox vaccine spread rapidly across Europe. In Spain, vaccinations first took place in December 1800 and the practice flourished upon the private initiative of doctors, surgeons, state officials and members of the nobility in different parts of the country. Ignacio María Ruiz de Luzuriaga, secretary of the Royal Academy of Medicine of Madrid, is considered in medical historiography as a key figure of the introduction of the smallpox vaccine in Spain. Ruiz de Luzuriaga had a major role as a disseminator of the Jennerian technique and as a distributor of the vaccine fluid. Given his prestige as a doctor and his position in the Royal Academy, he was commissioned to establish a scientific and academic corpus on preventive measures to foster their understanding, uptake and good practice among Spanish vaccinators. He also attempted to create a Central Vaccine Committee, such as that existing in other European countries. The Royal Academy kept records of his activity which have been filed and catalogued in a documentary set entitled 'Papeles sobre la vacuna' [Vaccine Papers]. This archive has not been studied in depth to date. These documents allow identifying a network of correspondents set up by Ruiz de Luzuriaga. He provided these correspondents with the vaccine and asked them to report back on the vaccination progress made in their municipalities. This correspondence provides an account of how the first immunisations in Spain unravelled, as well as of the initial concerns that accompanied the introduction of the vaccine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single chain variable fragments (scFvs) (SC34 and SC212) bound to vaccinia virus (VACV) from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to IC 50 values of 2 μg/mL (13.33 nM) and 22 μg/mL (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a PRNT 50 of ~0.16 mg/mL (~1.07 μM). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.

Vaccinia virus (VACV) is a notorious virus for a number of scientific reasons; however, most of its notoriety comes from the fact that it was used as a vaccine against smallpox, being ultimately responsible for the eradication of that disease. Nonetheless, many different vaccinia virus strains have been obtained over the years; some are suitable to be used as vaccines, whereas others are virulent and unsuitable for this purpose. Interestingly, different vaccinia virus strains elicit different immune responses in vivo , and this is a direct result of the genomic differences among strains. In order to evaluate the net result of virus-encoded immune evasion strategies of vaccinia viruses, we compared antiviral immune responses in mice intranasally infected by the highly attenuated and nonreplicative MVA strain, the attenuated and replicative Lister strain, or the virulent WR strain. Overall, cell responses elicited upon WR infections are downmodulated compared to those elicited by MVA and Lister infections, especially in determined cell compartments such as macrophages/monocytes and CD4 + T cells. CD4 + T cells are not only diminished in WR-infected mice but also less activated, as evaluated by the expression of costimulatory molecules such as CD25, CD212, and CD28 and by the production of cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-4 (IL-4), and IL-10. On the other hand, MVA infections are able to induce strong T-cell responses in mice, whereas Lister infections consistently induced responses that were intermediary between those induced by WR and MVA. Together, our results support a model in which the virulence of a VACV strain is proportional to its potential to downmodulate the host's immune responses. IMPORTANCE Vaccinia virus was used as vaccine against smallpox and was instrumental in the successful eradication of that disease. Although smallpox vaccination is no longer in place in the overall population, the use of vaccinia virus in the development of viral vector-based vaccines has become popular. Nonetheless, different vaccinia virus strains are known and induce different immune responses. To look into this, we compared immune responses triggered by mouse infections with the nonreplicative MVA strain, the attenuated Lister strain, or the virulent WR strain. We observed that the WR strain was capable of downmodulating mouse cell responses, whereas the highly attenuated MVA strain induced high levels of cell-mediated immunity. Infections by the intermediately attenuated Lister strain induced cell responses that were intermediary between those induced by WR and MVA. We propose that the virulence of a vaccinia virus strain is directly proportional to its ability to downmodulate specific compartments of antiviral cell responses., (Copyright © 2019 American Society for Microbiology.)

Since the National Smallpox Vaccination Program began in 2002, over 2.4 million military servicemembers have been inoculated. Use of bifurcated needles to introduce live vaccinia virus by repeated skin trauma is largely the same process that was first developed over 200 years ago, and is similar to that of tattoo administration. Both tattoos and smallpox inoculation can cause local immune dysregulation, with prior research cautioning further complications if they are placed in the same area within a close timeframe. Here we present the case of a soldier with smallpox autoinoculation, who reported receiving a tattoo directly over his inoculation site 4 days after vaccination. Initial scattered flesh-colored papules evolved over several days to classic umbilicated lesions with additional fevers, chills, myalgias, and suspected secondary infection. Thirteen cases of tattoo and smallpox inoculation complications have been reported in the Vaccine Adverse Events Reporting System (VAERS) database in 15 years. Current US Army Public Health Command (USAPHC) guidance mandates no new tattoos for 30 days post-inoculation, although the Military Vaccine Agency notes that this period may be extended to a window of greater than 60 days on a case-by-case basis. This incident illustrates the risks of current smallpox vaccination practice and poor patient adherence to post-vaccination care instructions. Continued use of traditional smallpox vaccination administration necessitates increased education and emphasis on proper inoculation site aftercare.

The review contains a brief analysis of the results of investigations conducted during 40 years after smallpox eradication and directed to study genomic organization and evolution of variola virus (VARV) and development of modern diagnostics, vaccines and chemotherapies of smallpox and other zoonotic orthopoxviral infections of humans. Taking into account that smallpox vaccination in several cases had adverse side effects, WHO recommended ceasing this vaccination after 1980 in all countries of the world. The result of this decision is that the mankind lost the collective immunity not only to smallpox, but also to other zoonotic orthopoxvirus infections. The ever more frequently recorded human cases of zoonotic orthopoxvirus infections force to renew consideration of the problem of possible smallpox reemergence resulting from natural evolution of these viruses. Analysis of the available archive data on smallpox epidemics, the history of ancient civilizations, and the newest data on the evolutionary relationship of orthopoxviruses has allowed us to hypothesize that VARV could have repeatedly reemerged via evolutionary changes in a zoonotic ancestor virus and then disappeared because of insufficient population size of isolated ancient civilizations. Only the historically last smallpox pandemic continued for a long time and was contained and stopped in the 20th century thanks to the joint efforts of medics and scientists from many countries under the aegis of WHO. Thus, there is no fundamental prohibition on potential reemergence of smallpox or a similar human disease in future in the course of natural evolution of the currently existing zoonotic orthopoxviruses. Correspondingly, it is of the utmost importance to develop and widely adopt state-of-the-art methods for efficient and rapid species-specific diagnosis of all orthopoxvirus species pathogenic for humans, VARV included. It is also most important to develop new safe methods for prevention and therapy of human orthopoxvirus infections., Competing Interests: The authors declare no conflict of interest.

"Immunogenic cell death" (ICD) is associated with the emission of so-called damage-associated molecular patterns (DAMPs) which trigger the immune response against dead-cell associated antigens. The secretion of the DAMP, adenosine triphosphate (ATP) has been shown to be autophagy-dependent. Here, we demonstrate that Modified Vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, induces both cell death and autophagy in murine bone marrow-derived dendritic cells (BMDCs), which in turn confer the (cross-)priming of OVA-specific cytotoxic T cells (OT-I cells). Additionally, we show that MVA infection leads to increased extracellular ATP (eATP) as well as intracellular ATP (iATP) levels, with the latter being influenced by the autophagy. Furthermore, we show that the increased eATP supports the proliferation of OT-I cells and inhibition of the P2RX7 receptors results in an abrogation of the proliferation. These data reveal novel mechanisms on how MVA enhances adaptive immunity in vaccine strategies., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Smallpox vaccinations were stopped globally in 1980. Recent studies have shown that in women, being smallpox vaccinated was associated with a reduced risk of HIV infection compared with not being smallpox vaccinated. At the initial infection, HIV-1 most often uses CCR5 as a co-receptor to infect the T-lymphocytes. We therefore investigated whether smallpox vaccination is associated with a down-regulation of CCR5 on the surface of peripheral T-lymphocytes in healthy women in Guinea-Bissau., Methods: We included HIV seronegative women from Bissau, Guinea-Bissau, born before 1974, with and without a smallpox vaccination scar. Blood samples were stabilised in a TransFix buffer solution and stained for flow cytometry according to a T-cell maturation profile., Results: Ninety-seven women were included in the study; 52 with a smallpox vaccination scar and 45 without a scar. No association between smallpox vaccination scar and CCR5 expression was found in any T-lymphocyte subtype., Conclusion: Among HIV seronegative women, being smallpox vaccinated more than 40 years ago was not associated with a down-regulation of CCR5 receptors on the surface of peripheral T-lymphocytes., Competing Interests: The authors have declared that no competing interests exist.

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design., (Copyright © 2018 by The American Association of Immunologists, Inc.)

We built a SEIR (susceptible, exposed, infected, recovered) model of smallpox transmission for New York, New York, USA, and Sydney, New South Wales, Australia, that accounted for age-specific population immunosuppression and residual vaccine immunity and conducted sensitivity analyses to estimate the effect these parameters might have on smallpox reemergence. At least 19% of New York's and 17% of Sydney's population are immunosuppressed. The highest smallpox infection rates were in persons 0-19 years of age, but the highest death rates were in those >45 years of age. Because of the low level of residual vaccine immunity, immunosuppression was more influential than vaccination on death and infection rates in our model. Despite widespread smallpox vaccination until 1980 in New York, smallpox outbreak severity appeared worse in New York than in Sydney. Immunosuppression is highly prevalent and should be considered in future smallpox outbreak models because excluding this factor probably underestimates death and infection rates.

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies., (Copyright © 2018. Published by Elsevier B.V.)

The increasing frequency of monkeypox virus infections, new outbreaks of other zoonotic orthopoxviruses and concern about the re-emergence of smallpox have prompted research into developing antiviral drugs and better vaccines against these viruses. This article considers the genetic engineering of vaccinia virus (VACV) to enhance vaccine immunogenicity and safety. The virulence, immunogenicity and protective efficacy of VACV strains engineered to lack specific immunomodulatory or host range proteins are described. The ultimate goal is to develop safer and more immunogenic VACV vaccines that induce long-lasting immunological memory., Competing Interests: The authors declare no conflict of interest.

The highly attenuated Modified Vaccinia virus Ankara (MVA) lacks most of the known vaccinia virus (VACV) virulence and immune evasion genes. Today MVA can serve as a safety-tested next-generation smallpox vaccine. Yet, we still need to learn about regulatory gene functions preserved in the MVA genome, such as the apoptosis inhibitor genes F1L and E3L . Here, we tested MVA vaccine preparations on the basis of the deletion mutant viruses MVA-ΔF1L and MVA-ΔE3L for efficacy against ectromelia virus (ECTV) challenge infections in mice. In non-permissive human tissue culture the MVA deletion mutant viruses produced reduced levels of the VACV envelope antigen B5. Upon mousepox challenge at three weeks after vaccination, MVA-ΔF1L and MVA-ΔE3L exhibited reduced protective capacity in comparison to wildtype MVA. Surprisingly, however, all vaccines proved equally protective against a lethal ECTV infection at two days after vaccination. Accordingly, the deletion mutant MVA vaccines induced high levels of virus-specific CD8+ T cells previously shown to be essential for rapidly protective MVA vaccination. These results suggest that inactivation of the anti-apoptotic genes F1L or E3L modulates the protective capacity of MVA vaccination most likely through the induction of distinct orthopoxvirus specific immunity in the absence of these viral regulatory proteins., Competing Interests: The authors declare no conflict of interest.

Background: Although smallpox was completely eliminated by 1980, it remains possible that variola virus could be intentionally released in an act of bioterrorism. Thus, several studies have been performed to detect antibody levels after smallpox vaccination of the current population in various countries to indicate the duration of maintenance of immunological memory. Our study endeavored to investigate the level of neutralizing (Nt) antibody responses of Thai individuals who had been immunized with smallpox vaccine during childhood., Methods: The plaque reduction neutralization test (PRNT) was used to study vaccinia Nt antibody responses in sera of individuals ranging in age from 35-4, 45-54, 55-64, 65-74, 75-84 and > 84 years old, referred to as groups 1-6, respectively. Each group included 200 sera: 100 male sera and 100 female sera., Results: An incubation time of 15 hours for sera and vaccinia virus was confirmed to be the optimal incubation period for PRNT. Positive Nt antibody titers (≥32) were detected in 135 (11.25%) of 1,200 sera: 81 (6.75%) male sera and 54 (4.5%) female sera. There were 4 (2%), 11 (5.5%), 19 (9.5%), 16 (8%), 33 (16.5%), and 52 (26%) positive sera in groups 1-6, respectively. Interestingly, the oldest individual with positive Nt antibody was a 98-year-old female. Two males aged 96 and 91 years old had the highest Nt antibody titers., Conclusions: Our data suggests that the vaccinia-specific Nt antibody response in the current Thai population could be maintained for more than 90 years after vaccination. However, the majority of the Thai population aged ≥35-74 years old is still highly susceptible to infection.

This article examines the biosecurity and biodefense implications resulting from the recent creation of horsepox virus, a noncirculating (extinct) species of orthopoxvirus. Here we examine the technical aspects of the horsepox virus synthesis and conclude that orthopox synthesis experiments currently remain technically challenging-and will continue to be so, even once this work is published in the scientific literature. This limits potential misuse by some types of nefarious actors. We also examine the implications of one stated purpose for the recreation of horsepox virus: the development of a smallpox vaccine. If the development is successful, it could take advantage of US government incentives for the priority FDA review of medical countermeasures (MCMs) against biosecurity threats. However, if this case leads to the determination that this incentive is counterproductive for security, the newly created priority review voucher program should be more clearly defined or limited based on need. Limiting the program could have costs that require further consideration, however, as general incentives for biodefense medical countermeasure development are required for MCMs to be available. Finally, while the recreation of horsepox virus was not technically trivial, nor was it cell-free, this experiment was a de facto demonstration of already-assumed scientific capabilities. The ability to recreate horsepox, or smallpox, will remain no matter what policy controls are put into place. It will be impossible to close off all avenues for nefarious misuse of gene synthesis, or misuse of biological materials more broadly. As a result, we advocate for the implementation of policy, regulations, and guidance that will make illicit recreation harder, more burdensome, more detectable, and, thus, more preventable without having sweeping negative consequences for the research enterprise. As part of our biosecurity efforts, we must also encourage and enable scientists to participate actively and to do all they can to safeguard their technical fields from irresponsible or illicit actions.

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 7days 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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years) and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity) may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb) responses to vaccinia waned after 5-10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT) of 250 to baseline (<20) after 10-20 years post vaccination. This contrasted with a comparator group of adults, ages 35-49, who were vaccinated with Dryvax® as children. In the childhood vaccinees, titers persisted for >30 years with a GMT of 210 (range 112-3234). This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program., Competing Interests: The authors have declared that no competing interests exist.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although many diseases and traits show large heritability, few genetic variants have been found to strongly separate phenotype groups by genotype. Complex regulatory networks of variants and expression of multiple genes lead to small individual-variant effects and difficulty replicating the effect of any single variant in an affected pathway. Interaction network modeling of GWAS identifies effects ignored by univariate models, but population differences may still cause specific genes to not replicate. Integrative network models may help detect indirect effects of variants in the underlying biological pathway. In this study, we used gene-level functional interaction information from the Integrative Multi-species Prediction (IMP) tool to reveal important genes associated with a complex phenotype through evidence from epistasis networks and pathway enrichment. We test this method for augmenting variant-based network analyses with functional interactions by applying it to a smallpox vaccine immune response GWAS. The integrative analysis spotlights the role of genes related to retinoid X receptor alpha (RXRA), which has been implicated in a previous epistasis network analysis of smallpox vaccine.

Expression quantitative trait loci (eQTL) studies have functionalized nucleic acid variants through the regulation of gene expression. Although most eQTL studies only examine the effects of single variants on transcription, a more complex process of variant-variant interaction (epistasis) may regulate transcription. Herein, we describe a tool called interaction QTL (iQTL) designed to efficiently detect epistatic interactions that regulate gene expression. To maximize biological relevance and minimize the computational and hypothesis testing burden, iQTL restricts interactions such that one variant is within a user-defined proximity of the transcript (cis-regulatory). We apply iQTL to a data set of 183 smallpox vaccine study participants with genome-wide association study and gene expression data from unstimulated samples and samples stimulated by inactivated vaccinia virus. While computing only 0.15% of possible interactions, we identify 11 probe sets whose expression is regulated through a variant-variant interaction. We highlight the functional epistatic interactions among apoptosis-related genes, DIABLO, TRAPPC4 and FADD, in the context of smallpox vaccination. We also use an integrative network approach to characterize these iQTL interactions in a posterior network of known prior functional interactions. iQTL is an efficient, open-source tool to analyze variant interactions in eQTL studies, providing better understanding of the function of epistasis in immune response and other complex phenotypes.

Objective: Smallpox vaccination has been associated with notable side effects and adverse events. This study assessed the frequency of each among public health workers immunized during the 2003 Arkansas civilian smallpox vaccination campaign to allow individuals and policymakers to make informed decisions whether repeat vaccination, as recommended in 10-year intervals, should be considered., Design and Sample: This descriptive study summarizes postvaccination surveillance data for all civilians receiving smallpox vaccine (Dryvax) in Arkansas in 2003., Measures: Rates of side effects and adverse events were determined. Vaccinia-specific antibody titers among a subset of public health response team members were also assessed., Results: Of the 1,124 vaccine recipients, 87% had a major take response. Substantial symptomatology, a 2% adverse event rate, a 0.5% hospitalization rate, and zero inadvertent transmission following vaccination were observed. Vaccinia-specific antibody titers increased on average 9-fold from 2.21*10(2) to 2.16*10(3) one month after vaccination. We found no association of age, sex, or racial subgroups with adverse events, hospitalizations, a lower take response rate, or lower postvaccination antibody titers., Conclusions: Prominent side effect profiles and adverse events among study participants seem to support individual and institutional reluctance to vaccinate civilians in the absence of smallpox reemergence., (© 2015 Wiley Periodicals, Inc.)

Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

Ectromelia virus is a mouse-specific orthopoxvirus that, following footpad infection or natural transmission, causes mousepox in most strains of mice, while a few strains, such as C57BL/6, are resistant to the disease but not to the infection. Mousepox is an acute, systemic, highly lethal disease of remarkable semblance to smallpox, caused by the human-specific variola virus. Starting in 1929 with its discovery by Marchal, work with ECTV has provided essential information for our current understanding on how viruses spread lympho-hematogenously, the genetic control of antiviral resistance, the role of different components of the innate and adaptive immune system in the control of primary and secondary infections with acute viruses, and how the mechanisms of immune evasion deployed by the virus affect virulence in vivo. Here, I review the literature on the pathogenesis and immunobiology of ECTV infection in vivo., (© 2016 Elsevier Inc. All rights reserved.)

Background: Attenuated vaccinia virus strain, LC16m8, defective in the B5R envelope protein gene, is used as a stockpile smallpox vaccine strain in Japan against bioterrorism: the defect in the B5R gene mainly contributes to its highly attenuated properties., Methods: The protective activity of LC16m8 vaccine against challenge with a lethal dose of vaccinia Western Reserve strain was assessed in wild-type and immunodeficient mice lacking CD4, MHC class I, MHC class II or MHC class I and II antigens., Results: The immunization with LC16m8 induced strong protective activity comparable to that of its parent strain, Lister (Elstree) strain, in wild-type mice from 2 days to 1 year after vaccination, as well as in immunodeficient mice at 2 or 3 weeks after vaccination. These results implicated that the defect in the B5R gene hardly affected the potential activity of LC16m8 to induce innate, cell-mediated and humoral immunity, and that LC16m8 could be effective in immunodeficient patients., Conclusion: LC16m8 with truncated B5 protein has an activity to induce immunity, such as innate immunity and subsequent cell-mediated and humoral immunity almost completely comparable to the activity of its parental strain Lister., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Published by Elsevier Ltd.)

Background: The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein., Methods: We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation., Results: The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections., Conclusion: These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.

Vaccines are the most successful strategy developed in Medicine to prevent and even eradicate the most dreadful epidemic infectious diseases. The history of smallpox vaccination in Naples is quite unique. Although Galbiati established the retro-vaccination (1803) and developed the "calf" lymph vaccine, recognized and implemented since 1864 as the optimal smallpox vaccine in the following hundred years, Naples general population was mainly vaccinated with "human" lymph from abandoned children until 1893. Mini-epidemics of syphilis and serum hepatitis were periodically reported as results of arm-to-arm procedure. The risk of transmission of blood-related pathogens was higher in Naples where >80% of abandoned children, used as repository of cowpox virus, were dying in their first year of life. Recent vaccinology standards finally eliminated the risk of adventitious contaminating pathogens. Implementation of hepatitis B vaccination since 1991 eventually contributed to current HBV prevalence in Campania region <1%, within the range of the European Countries.