Brazilian vaccinia viruses and their origins

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

A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.     

Surveillance of orthopoxvirus infections, and associated research, in the period after smallpox eradication

In 1980, the World Health Assembly declared the global eradication of smallpox and recommended the universal discontinuation of smallpox vaccination; nevertheless, it recommended that surveillance and research on orthopoxvirus infections should continue. By early 1982, all except 8 countries in the world had stopped routine vaccination programmes and all except 1 no longer required an international certificate of smallpox vaccination for travellers. Since 1978, as a result of continuing active surveillance, 176 smallpox rumours have been investigated in 60 countries. Two of these concerned the two laboratory-associated cases that occurred in the United Kingdom in 1978; all the others were false alarms. Special surveillance programmes for human monkeypox have been developed in West and Central Africa. The number of laboratories retaining variola virus stocks has been reduced to four. Investigations to determine the identity and origin of the six known isolates of “whitepox” virus have continued. Research on mapping of variola DNA and on monoclonal antibodies against certain orthopoxvirus antigens is continuing. All these measures are aimed at ensuring that the achievement of smallpox eradication is permanent.     

Fatal Cowpox Virus Infection in Human Fetus,France, 2017

Cowpox virus (CPXV) has an animal reservoir and is typically transmitted to humans by contact with infected animals. In 2017, CPXV infection of a pregnant woman in France led to the death of her fetus. Fetal death after maternal orthopoxvirus (smallpox) vaccination has been reported; however, this patient had not been vaccinated. Investigation of the patient’s domestic animals failed to demonstrate prevalence of CPXV infection among them. The patient’s diagnosis was confirmed by identifying CPXV DNA in all fetal and maternal biopsy samples and infectious CPXV in biopsy but not plasma samples. This case of fetal death highlights the risk for complications of orthopoxvirus infection during pregnancy. Among orthopoxviruses, fetal infection has been reported for variola virus and vaccinia virus; our findings suggest that CPXV poses the same threats for infection complications as vaccinia virus.     

Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies

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

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

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

Laboratory-acquired West Nile virus infections--United States, 2002

West Nile virus (WNV), a mosquito-borne flavivirus introduced recently to North America, is a human, equine, and avian neuropathogen. The majority of human infections with WNV are mosquito-borne; however, laboratory-acquired infections with WNV and other arboviruses also occur. This report summarizes two recent cases of WNV infection in laboratory workers without other known risk factors who acquired infection through percutaneous inoculation. Laboratory workers handling fluids or tissues known or suspected to be WNV-infected should minimize their risk for exposure and should report injuries and illnesses of suspected occupational origin to their supervisor.     

Emergence and reemergence of smallpox: the need for development of a new generation smallpox vaccine

The review summarizes the archive data on smallpox, history of ancient civilizations, and the most recent data on the genome organization of orthopoxviruses, their evolutionary relationships, and the time points of smallpox emergence. The performed analysis provides the grounds for the hypothesis that smallpox could have emerged several times as a result of evolutionary changes in the zoonotic ancestor virus and disappeared due to insufficient population size of ancient civilizations. Smallpox reemerged in the Indian subcontinent approximately 2500-3000 years before present, which resulted in endemization of this anthroponotic infection, which had been preserved until the smallpox eradication in the 20th century AD. The conclusion suggests a potential possibility of future variola virus reemergence, presenting a great menace for mankind, as well as the need for development of new safe smallpox vaccines, design of anti-smallpox drugs, and activation of the control of zoonotic human orthopoxvirus infections.     

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 10⁶ pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.     

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

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

Retrospective analysis of monkeypox infection

Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks. Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections. We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections. Despite the difficulty of diagnosing clinically inapparent monkeypox in previously vaccinated persons, this technique exhibited 100% sensitivity and 100% specificity for identifying clinically overt monkeypox infection irrespective of vaccination history. We also describe a Western blot technique in which up to 3 diagnostic bands may be used to distinguish between vaccinia and monkeypox infection. The techniques described provide independent diagnostic tests suitable for retrospective analysis of monkeypox outbreaks.     

Accidental infection of laboratory worker with vaccinia virus

We report the accidental needlestick inoculation of a laboratory worker with vaccinia virus. Although the patient had previously been vaccinated against smallpox, severe lesions appeared on the fingers. Western blot and polymerase chain reaction-restriction fragment length polymorphism were used to analyze the virus recovered from the lesions. The vaccinia virus-specific immunoglobulin G levels were measured by enzyme-linked immunosorbent assay. Our study supports the need for vaccination for laboratory workers that routinely handle orthopoxvirus.     

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

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

Smallpox and its post-eradication surveillance

Since May 1980 when the 33rd World Health Assembly declared the global eradication of smallpox, WHO has been developing a comprehensive system of surveillance aimed at maintaining the world permanently free from this disease. By 1984, all countries had ceased vaccinating the general public against smallpox, and had withdrawn the requirement for smallpox vaccination certificates from international travellers. A number of countries had also discontinued the vaccinating of military personnel. Until now WHO has maintained a stock of smallpox vaccine sufficient to vaccinate 300 million persons, but considering that 10 years have elapsed since the last endemic case of smallpox, maintenance of this reserve is no longer indicated. WHO continues to monitor rumors and coordinate the investigation of suspected cases, all of which have actually been misdiagnosed chickenpox or some other skin disease, or other errors in recording or reporting. Variola virus is now kept in only 2 WHO Collaborating Centers which possess high security containment laboratories. Since the variola virus gene pool has been cloned in bacterial plasmids which provide sufficient material to solve future research and diagnostic problems, there is no need to retain stocks of viable variola virus any longer. The results of a special program for the surveillance of human monkeypox have confirmed that the disease does not pose any significant health problem. In addition to testing human and animal specimens, WHO collaborating laboratories have made progress in the analysis of DNA of orthopoxviruses and in the development of reliable serological tests.     

Characterization of antibodies to orthopoxviruses in human sera by radioimmunoassay

Serological surveillance of suspected orthopoxvirus infections in man is important for confirming the success of the worldwide smallpox eradication programme. An adsorption radioimmunoassay (RIA) was used to differentiate sera from patients who were naturally infected with human monkeypox or variola virus, and individuals who were immunized with vaccinia virus. The antisera were adsorbed with uninfected chicken chorioallantoic membrane (CAM) and vaccinia-infected CAM before reacting in RIA with vaccinia, monkeypox, and variola antigens. Each serum group showed characteristic patterns of residual antibody activity which made it possible to identify antibody specificities.     

Cross-sectional study involving healthcare professionals in a Vaccinia virus endemic area

Orthopoxviruses (OPV) are emerging viruses with great importance in human and veterinary medicine, such as Vaccinia virus (VACV), which causes outbreaks of bovine vaccinia (BV) in South America. The clinical aspects of BV are similar to other vesicular infections, complicating the clinical diagnosis. This cross-sectional study evaluated the knowledge of Healthcare Professionals about BV and revealed their unpreparedness about BV in a VACV hyper-endemic area in Brazil, highlighting the public health issues associated with VACV infections. This study presents an opportunity to discuss the importance of vaccination for healthcare professionals who work in areas of VACV circulation and brings an educational measure on VACV infections for health professionals around the world.     

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

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

Update: cardiac and other adverse events following civilian smallpox vaccination--United States, 2003

During January 24-June 20, 2003, smallpox vaccine was administered to 37,802 civilian health-care and public health workers in 55 jurisdictions to prepare the United States for a possible terrorist attack using smallpox virus. This report updates information on vaccine-associated adverse events among civilians vaccinated since the beginning of the program and among contacts of vaccinees, received by CDC from the Vaccine Adverse Event Reporting System (VAERS) as of June 20. Two cases of dilated cardiomyopathy (DCM) were diagnosed 3 months after vaccination. For the potential relation between smallpox vaccine and DCM to be assessed, identification of additional cases of DCM among vaccinees will be essential. Physicians who treat smallpox vaccine recipients are encouraged to evaluate and report patients with symptoms compatible with DCM, including those that occur several months after vaccination.     

Developing new smallpox vaccines.

New stockpiles of smallpox vaccine are required as a contingency for protecting civilian and military personnel against deliberate dissemination of smallpox virus by terrorists or unfriendly governments. The smallpox vaccine in the current stockpile consists of a live animal poxvirus (Vaccinia virus [VACV]) that was grown on the skin of calves. Because of potential issues with controlling this earlier manufacturing process, which included scraping VACV lesions from calfskin, new vaccines are being developed and manufactured by using viral propagation on well-characterized cell substrates. We describe, from a regulatory perspective, the various strains of VACV, the adverse events associated with calf lymph-propagated smallpox vaccine, the issues regarding selection and use of cell substrates for vaccine production, and the issues involved in demonstrating evidence of safety and efficacy.     

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

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