Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.     

Assessment of the Protective Effect of Imvamune and Acam2000 Vaccines against Aerosolized Monkeypox Virus in Cynomolgus Macaques

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.     

Immunization of mice with vaccinia virus Tiantan strain yields antibodies cross-reactive with protective antigens of monkeypox virus

Highlights
1. The first study describing the cross-reactivity of antibodies elicited by a Chinese smallpox vaccine against monkeypox virus.
2. Mice immunized with vaccinia virus Tiantan strain yield antibodies cross-reactive with monkeypox virus protective antigens.
3. Cross-reactivities of VTT-elicited antibodies against monkeypox protective antigens are ranging from 33% to 94%.     

Outbreaks of human monkeypox after cessation of smallpox vaccination

The recent observation of a surge in human monkeypox in the Democratic Republic of the Congo (DRC) prompts the question of whether cessation of smallpox vaccination is driving the phenomenon, and if so, why is re-emergence not universal throughout the historic geographic range of the virus? Research addressing the virus's mechanisms for immune evasion and induction, as well as that directed at elucidating the genes involved in pathogenesis in different viral lineages (West African vs Congo Basin), provide insights to help explain why emergence appears to be geographically limited. Novel vaccines offer one solution to curtail the spread of this disease.     

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.     

Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).     

Insights into human CD8(+) T-cell memory using the yellow fever and smallpox vaccines

Live virus vaccines provide a unique opportunity to study human CD8(+) T-cell memory in the context of a controlled, primary acute viral infection. Yellow fever virus-17D and Dryvax are two such live-virus vaccines that are highly efficacious, used worldwide and provide long-term immunity against yellow fever and smallpox respectively. In this review, we describe the properties of virus-specific memory CD8(+) T cells generated in smallpox and yellow fever vaccinees. We address fundamental questions regarding magnitude, functional quality and longevity of the CD8(+) T-cell response, which are otherwise challenging to address in humans. These findings provide insights into the attributes of the human immune system as well as provide a benchmark for the optimal quality of a CD8(+) T-cell response that can be used to evaluate novel candidate vaccines.     

CD8 T cells are essential for recovery from a respiratory vaccinia virus infection

The precise immune components required for protection against a respiratory Orthopoxvirus infection, such as human smallpox or monkeypox, remain to be fully identified. In this study, we used the virulent Western Reserve strain of vaccinia virus (VACV-WR) to model a primary respiratory Orthopoxvirus infection. Naive mice infected with VACV-WR mounted an early CD8 T cell response directed against dominant and subdominant VACV-WR Ags, followed by a CD4 T cell and Ig response. In contrast to other VACV-WR infection models that highlight the critical requirement for CD4 T cells and Ig, we found that only mice deficient in CD8 T cells presented with severe cachexia, pulmonary inflammation, viral dissemination, and 100% mortality. Depletion of CD8 T cells at specified times throughout infection highlighted that they perform their critical function between days 4 and 6 postinfection and that their protective requirement is critically dictated by initial viral load and virulence. Finally, the ability of adoptively transferred naive CD8 T cells to protect RAG(-/-) mice against a lethal VACV-WR infection demonstrated that they are both necessary and sufficient in protecting against a primary VACV-WR infection of the respiratory tract.     

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.     

猴痘、天花病毒感染快速分子诊断荧光定量实时PCR方法的建立

目的:天花、猴痘可感染人并引起严重皮疹、发热等临床症状,均为烈性传染病,是潜在的生物恐怖因子,因此需要建立针对其感染的快速特异的诊断方法。方法:分别设计正痘病毒属通用型、天花病毒特异、猴痘病毒特异的引物与荧光标记探针,建立荧光定量实时PCR方法,对人工合成或模拟样本进行检测。结果:可在4h内对天花或猴痘病毒感染进行特异性鉴别诊断,检测灵敏度可达100拷贝/25μL反应体积。结论:本方法可作为一种检疫与反恐应急储备技术。     

Coadministration of Cidofovir and Smallpox Vaccine Reduced Vaccination Side Effects but Interfered with Vaccine-Elicited Immune Responses and Immunity to Monkeypox

While the smallpox vaccine, Dryvax or Dryvax-derived ACAM2000, holds potential for public immunization against the spread of smallpox by bioterror, there is serious concern about Dryvax-mediated side effects. Here, we report that a single-dose vaccination regimen comprised of Dryvax and an antiviral agent, cidofovir, could reduce vaccinia viral loads after vaccination and significantly control Dryvax vaccination side effects. However, coadministration of cidofovir and Dryvax also reduced vaccine-elicited immune responses of antibody and T effector cells despite the fact that the reduced priming could be boosted as a recall response after monkeypox virus challenge. Evaluations of four different aspects of vaccine efficacy showed that coadministration of cidofovir and Dryvax compromised the Dryvax-induced immunity against monkeypox, although the covaccinated monkeys exhibited measurable protection against monkeypox compared to that of naïve controls. Thus, the single-dose coadministration of cidofovir and Dryvax effectively controlled vaccination side effects but significantly compromised vaccine-elicited immune responses and vaccine-induced immunity to monkeypox.     

Prospects for new viral vaccines

Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals. Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels. Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B. The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens. Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development. The impact of recent experiments in cloning hepatitis B virus DNA in E. coli on the production of a purified viral polypeptide vaccine is assessed.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

Correlates of protective immunity in poxvirus infection: where does antibody stand?

Even though smallpox has been eradicated, the threat of accidental or intentional release has highlighted the fact there is little consensus about correlates of protective immunity or immunity against re-infection with the causative poxvirus, variola virus (VARV). As the existing vaccine for smallpox has unacceptable rates of side effects and complications, new vaccines are urgently needed. Surrogate animal models of VARV infection in humans, including vaccinia virus (VACV) and ectromelia virus (ECTV) infection in mice, monkeypox virus (MPXV) infection in macaques have been used as tools to dissect the immune response to poxviruses. Mousepox, caused by ECTV, a natural mouse pathogen, is arguably the best surrogate small-animal model, as it shares many aspects of virus biology, pathology and clinical features with smallpox in humans. The requirements for recovery from a primary ECTV infection have been well characterized and include type I and II interferons, natural killer cells, CD4T cells, CD8T cell effector function and antibody. From a vaccine standpoint, it is imperative that the requirements for recovery from secondary infection are also identified. We have investigated host immune parameters in response to a secondary ECTV infection, and have identified that interferon and CD8T cell effector functions are not essential; however, T- and B-cell interaction and antibody are absolutely critical for recovery from a secondary challenge. The central role of antibody has been also been identified in the secondary response to other poxviruses. These findings have important clinical implications and would greatly assist the design of therapeutic interventions and new vaccines for smallpox.     

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.     

猴痘的流行现状及防控

猴痘(monkeypox)是由猴痘病毒感染所致的人兽共患病,主要发生在非洲中部、西部地区。猴痘病毒可感染多种哺乳类动物,主要在动物中流行,人接触感染动物后可被传染。猴痘的临床表现与天花相似(发热、皮疹等),但症状较轻。天花疫苗接种可提供预防猴痘的免疫保护力。然而,因全球天花被消灭而停止接种天花疫苗后,猴痘成为最可能威胁人类的正痘病毒性疾病。近期,其散发病例在欧洲多地出现。2022年5月7日英国报道了猴痘疫情。随后,欧洲报道猴痘确诊和疑似病例超过100例。猴痘主要传播途径包括接触感染动物、与患者直接接触或间接接触。2022年5月20日,世界卫生组织就此次猴痘疫情召开了紧急会议,旨在提高对猴痘的认识,做好防范应对准备。世界卫生组织、美国疾病预防控制中心、英国卫生部门报告了相关疫情并制定了相应的防控措施。截至2022年5月28日我国尚无输入性猴痘报道,但因国际交往频繁等仍须提高警惕。本文介绍了猴痘流行现状及有关防控信息,以供借鉴。     

Progress for dengue virus diseases. Towards the NS2B-NS3pro inhibition for a therapeutic-based approach

Transmitted by the Aedes aegypti mosquito, the dengue virus is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, and, as such, is a significant factor in the high death rate found in most tropical and subtropical areas of the world. Dengue diseases are not only a health burden to developing countries, but pose an emerging problem worldwide. The immunopathological mechanisms appear to include a complex series of immune responses. A rapid increase in the levels of cytokines and chemical mediators during dengue disease plays a key role in inducing plasma leakage, shock and hemorrhagic manifestations. Currently, there are no vaccines available against dengue virus, although several tetravalent live-attenuated dengue vaccines are in clinical phases I or II, and prevention through vaccination has become a major priority on the agendas of the World Health Organization and of national ministries of health and military organizations. An alternative to vaccines is found in therapeutic-based approaches. Understanding the molecular mechanisms of viral replication has led to the development of potential drugs, and new molecular viral targets for therapy are emerging. The NS3 protease domain of the NS3 protein is responsible for processing the viral polyprotein and its inhibition is one of the principal aims of pharmacological therapy. This review is an overview of the progress made against dengue virus; in particular, it examines the unique properties--structural and functional--of the NS3 protease for the treatment of dengue virus infections by the inhibition of viral polyprotein processing.     

Immunopathogenesis of poxvirus infections: forecasting the impending storm

Variola virus, the causative agent of smallpox, is a member of the poxvirus family and one of the most virulent human pathogens known. Although smallpox was eradicated almost 30 years ago, it is not understood why the mortality rates associated with the disease were high, why some patients recovered, and what constitutes an effective host response against infection. As variola virus infects only humans, our current understanding of poxvirus infections comes largely from historical clinical data from smallpox patients and from animal studies using closely related viruses such as ectromelia, myxoma and monkeypox. The outcome of an infection is determined by a complex interaction between the type of immune response mounted by the host and by evasion mechanisms that the virus has evolved to subvert it. Disease pathogenesis is also a function of both host and viral factors. Poxviruses are not only cytopathic, causing host tissue damage, but also encode an array of immunomodulatory molecules that affect the severity of disease. The ability of the host to control virus replication is therefore critical in limiting tissue damage. However, in addition to targeting virus, the immune response can inadvertently damage the host to such a degree that it causes illness and even death. There is growing evidence that many of the symptoms associated with serious poxvirus infections are a result of a 'cytokine storm' or sepsis and that this may be the underlying cause of pathology.