Replication-competent Vaccinia Virus Research Articles

Poxviridae Pneumonia.

Poxviridae family includes several viruses that infecting humans usually causes skin lesions only, but in some cases their clinical course is complicated by viral pneumonia (with or without bacterial superinfections). Historically variola virus has been the poxviridae most frequently associated with the development of pneumonia with many large outbreaks worldwide before its eradication in 1980. It is still considered a biological threat for its potential in biological warfare and bioterrorism. Smallpox pneumonia can be severe with the onset of acute respiratory distress syndrome (ARDS) and death. Vaccinia virus, used for vaccination against smallpox exceptionally, in immunocompromised patients, can induce generalized (with also lung involvement) severe disease after vaccination. MPXV virus occasionally can cause pneumonia particularly in immunocompromised patients. The pathophysiology of poxviridae pneumonia is still an area of active research; however, in animal models these viruses can cause both direct damage to the lower airways epithelium and a hyperinflammatory syndrome, like a cytokine storm. Multiple mechanisms of immune evasion have also been described. The treatment of poxviridae pneumonia is mainly based on careful supportive care. Despite the absence of randomized clinical trials in patients with poxviridae pneumonia there are antiviral drugs, such as tecovirimat, cidofovir and brincidofovir, FDA-approved for use in smallpox and also available under an expanded access protocol for treatment of MPXV. There are 2 (replication-deficient modified vaccinia Ankara and replication-competent vaccinia virus) smallpox vaccines FDA-approved with the first one also approved for prevention of MPXV in adults that are at high risk of infection.

Global Attention to Monkeypox

Monkeypox is brought on by a pox virus that is closely linked to the smallpox virus and is spread by intimate contact between humans and animals. When people come into contact with sick animals, they may unintentionally contract the monkeypox. Smallpox vaccinations cross-protect against MPXV due to antigenic similarities. Fever, headache, muscle aches, lymphadenopathy, and a recognisable rash that later develops into papules, vesicles, and pustules that scab over and recover are all signs of monkeypox (MPX). There are now two vaccines on the market: JYNNEOSTM (live, replication incompetent vaccinia virus) and ACAM2000® (live, replication competent vaccinia virus). Antivirals (such as tecovirimat, brincidofovir, and cidofovir) and vaccinia immune globulin intravenous (VIGIV) are available as therapies for monkeypox, albeit the majority of cases will have moderate and self-limited disease and supportive care is often sufficient. The ongoing outbreak, which has more than 10,000 cases in more than 50 nations between May and July 2022, illustrates how easily MPXV can spread among people and how this could pose a serious threat to public health with worldwide repercussions.

Monkey pox: A public health emergency.

Human monkeypox virus, a double-stranded DNA virus belongs to the Orthopoxvirus genus of the Poxviridae family with two genetic clades identified as West African and Central African [1]. First detected in 1958 when there was an outbreak of a vesicular disease among monkeys held in captivity being transported from Africa to Copenhagen, Denmark for research. The first case of monkeypox in humans was seen in a 9-year-old child belonging to the rural area of Zaire in August 1970, having smallpox-like vesicular lesions on the skin [1]. There have been reports of imported sporadic cases in Singapore, the UK, and the US from 2018 until recently in 2022, with the ongoing COVID-19 pandemic [2], an outbreak of monkeypox cases in the UK, other European and non-European countries was reported with confirmed cases of 2677 up to June 21, 2022 [3]. It is still not understood how the virus is transmitted to humans. It is assumed that the primary source of infection transmission is from animals to humans by handling monkeypox-infected animals. Secondary human-human transmission occurs through large respiratory droplets, contact with bodily fluids, lesions, and polluted surfaces such as clothing or linens [1,4]. Currently, many cases in the ongoing outbreak have been traced to sexual transmission, particularly among men who identify as gay or bisexual [5]. Initial symptoms begin with fever, headache, fatigue, and lymphadenopathy. Mucosal lesions in the mouth followed by lesions of skin on the face and extremities develop after 1–2 days. The lesions may vary in number from a small amount to thousands and may or may not spread to other parts of the body [6]. However, monkeypox cases reported in the current ongoing outbreak are atypical in nature with the characteristic rash developing in the genital and perianal areas with or without spreading to other body parts [7]. Even though, there is no specific treatment for monkeypox, the smallpox antivirals such as cidofovir, brincidofovir, and tecovirimat show activity against monkeypox with the latter two drugs having US FDA approval for use in treating smallpox. Most likely these medications would be reserved for treating severe cases or immunocompromised individuals and can be obtained through a public health department or the CDC [8]. The smallpox vaccines are believed to be effective in preventing monkeypox and as postexposure prophylaxis. JYNNEOS, which is a new generation smallpox vaccine, has an FDA indication for preventing monkeypox, and the older generation ACAM2000 used off-label can be used as well. Immediate post-exposure administration of the prophylactic vaccine can avoid infection or significantly reduce it. Alternative postexposure prophylaxis is vaccinia immune globulin in cases where the smallpox vaccine is contraindicated [8]. JYNNEOS vaccine can be administered in two doses, 28 days apart [9], and ACAM2000, which is a replication-competent live vaccinia virus vaccine can be administered as a single dose [10]. In conclusion, with the ongoing COVID-19 pandemic, monkeypox poses a great threat and has the potential to become a pandemic but with proper preventive strategies, the chances of containment of this disease increase to a great extent. Monkeypox, like most other viral infections, has symptomatic treatment. Educating patients and healthcare workers in areas where this disease is endemic is of utmost importance. People with knowledge about monkeypox can practice social distancing and other preventive strategies such as wearing a mask, washing hands regularly, and practicing safe sex. Healthcare workers with adequate knowledge about this disease can devise treatment plans and diagnostic strategies to help curb this virus among the masses. The infected individual must remain in isolation, wear a surgical mask, and keep the lesions covered as much as reasonably possible until all lesion crusts have naturally fallen off and a new skin layer has formed. Hospitals in endemic areas must acquire vaccines and medicines in time and make them available to the o masses. Government should encourage people to get vaccinated and educate and aware the general population about this through numerous social media platforms including TV, social media platforms, and messages on mobile phones. In areas where access to these things is lesser, mass campaigns need to be organized and people need to be taught in simple language, easy to understand. Ethical approval N/A. Sources of funding I declare no financial or competing interests. Author contributions Ahmed Kamal Siddiqi: Study concept and design. Umeed Khan: Acquisition of the data and drafting of the manuscript. Muhammad Adil Obaid: Acquisition of the data and drafting of the manuscript. Conflicts of interest I declare no financial or competing interests. Registration of research studies Name of the registry: N/A/ Unique Identifying number or registration ID: N/A. Hyperlink to your specific registration (must be publicly accessible and will be checked): N/A. Guarantor Ahmed Kamal Siddiqi [email protected] +92–03121222808. Consent N/A.

Adeno-associated virus-based malaria booster vaccine following attenuated replication-competent vaccinia virus LC16m8Δ priming

We previously demonstrated that boosting with adeno-associated virus (AAV) type 1 (AAV1) can induce highly effective and long-lasting protective immune responses against malaria parasites when combined with replication-deficient adenovirus priming in a rodent model. In the present study, we compared the efficacy of two different AAV serotypes, AAV1 and AAV5, as malaria booster vaccines following priming with the attenuated replication-competent vaccinia virus strain LC16m8Δ (m8Δ), which harbors the fusion gene encoding both the pre-erythrocytic stage protein, Plasmodium falciparum circumsporozoite (PfCSP) and the sexual stage protein (Pfs25) in a two-dose heterologous prime-boost immunization regimen. Both regimens, m8Δ/AAV1 and m8Δ/AAV5, induced robust anti-PfCSP and anti-Pfs25 antibodies. To evaluate the protective efficacy, the mice were challenged with sporozoites twice after immunization. At the first sporozoite challenge, m8Δ/AAV5 achieved 100% sterile protection whereas m8Δ/AAV1 achieved 70% protection. However, at the second challenge, 100% of the surviving mice from the first challenge were protected in the m8Δ/AAV1 group whereas only 55.6% of those in the m8Δ/AAV5 group were protected. Regarding the transmission-blocking efficacy, we found that both immunization regimens induced high levels of transmission-reducing activity (>99%) and transmission-blocking activity (>95%). Our data indicate that the AAV5-based multistage malaria vaccine is as effective as the AAV1-based vaccine when administered following an m8Δ-based vaccine. These results suggest that AAV5 could be a viable alternate vaccine vector as a malaria booster vaccine.

Intranasal Immunization with a Vaccinia Virus Vaccine Vector Expressing Pre-Fusion Stabilized SARS-CoV-2 Spike Fully Protected Mice against Lethal Challenge with the Heavily Mutated Mouse-Adapted SARS2-N501YMA30 Strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated as a variant of concern because its spike protein is heavily mutated. In particular, the Omicron spike is mutated at five positions (K417, N440, E484, Q493, and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at four of the five positions in the Omicron spike associated with neutralizing antibody escape (K417, E484, Q493, and N501). In this manuscript, we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against symptoms and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that the Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus—administered without parenteral injection—can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.

Prevention and Treatment of Monkeypox.

Human monkeypox is a zoonotic orthopoxvirus with presentation similar to smallpox. Monkeypox is transmitted incidentally to humans when they encounter infected animals. Reports have shown that the virus can also be transmitted through direct contact (sexual or skin-to-skin), respiratory droplets, and via fomites such as towels and bedding. Multiple medical countermeasures are stockpiled for orthopoxviruses such as monkeypox. Two vaccines are currently available, JYNNEOSTM (live, replication incompetent vaccinia virus) and ACAM2000® (live, replication competent vaccinia virus). While most cases of monkeypox will have mild and self-limited disease, with supportive care being typically sufficient, antivirals (e.g. tecovirimat, brincidofovir, cidofovir) and vaccinia immune globulin intravenous (VIGIV) are available as treatments. Antivirals can be considered in severe disease, immunocompromised patients, pediatrics, pregnant and breastfeeding women, complicated lesions, and when lesions appear near the mouth, eyes, and genitals. The purpose of this short review is to describe each of these countermeasures.

Vaccinia Virus Infection Inhibits Skin Dendritic Cell Migration to the Draining Lymph Node.

There is a paucity of information on dendritic cell (DC) responses to vaccinia virus (VACV), including the traffic of DCs to the draining lymph node (dLN). In this study, using a mouse model of infection, we studied skin DC migration in response to VACV and compared it with the tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG), another live attenuated vaccine administered via the skin. In stark contrast to BCG, skin DCs did not relocate to the dLN in response to VACV. Infection with UV-inactivated VACV or modified VACV Ankara promoted DC movement to the dLN, indicating that interference with skin DC migration requires replication-competent VACV. This suppressive effect of VACV was capable of mitigating responses to a secondary challenge with BCG in the skin, ablating DC migration, reducing BCG transport, and delaying CD4+ T cell priming in the dLN. Expression of inflammatory mediators associated with BCG-triggered DC migration were absent from virus-injected skin, suggesting that other pathways invoke DC movement in response to replication-deficient VACV. Despite adamant suppression of DC migration, VACV was still detected early in the dLN and primed Ag-specific CD4+ T cells. In summary, VACV blocks skin DC mobilization from the site of infection while retaining the ability to access the dLN to prime CD4+ T cells.

Replication-inducible vaccinia virus vectors with enhanced safety in vivo.

Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.

Improved immune response against HIV-1 Env antigen by enhancing EEV production via a K151E mutation in the A34R gene of replication-competent vaccinia virus Tiantan

The development of an effective HIV-1 vaccine is still a global priority. In recent years, vaccinia virus (VV) has been widely used as an HIV-1 vaccine vector, but its immune efficacy against HIV-1 antigens needs to be optimized. The extracellular enveloped virus (EEV) of VV is capable of faster entry, earlier release, and long-range dissemination. We hypothesized that an improvement in EEV formation by the manipulation of VV genes involved in the EEV release would consequently cause an improved expression of the VV carrying HIV-1 Env antigen and a subsequent enhanced immune response. To this end, an A34R K151E mutant (rVTT-A34Rmut) from VV Tiantan strain (VTT) with robustly increased EEV release was selected to serve as an optimized vaccine vector. The results were consistent with our hypothesis: the A34R mutant-based HIV-1 vaccine candidate rVTT-A34Rmut-Env produced more HIV-1 Env antigen in vitro and in vivo, and thus led to an improved HIV-1 Env-specific T cell immune response, binding antibody, and even the neutralizing antibody response in mice without increased virulence. Meanwhile, the application of the A34R mutation on another VV-based HIV-1 vaccine candidate, VTKgpe, also exhibited a similar immune enhancement effect with no enhanced virulence. The results in this study suggested that rVTT-A34Rmut is a potentially improved vaccine vector candidate for human application. In addition, the improvement of the EEV formation via the A34R gene mutation may also be potent in other poxvirus vector-based vaccines against HIV-1 or other pathogens and even cancer in the future.

Amplification of Oncolytic Vaccinia Virus Widespread Tumor Cell Killing by Sunitinib through Multiple Mechanisms.

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8+ T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8+ T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8+ T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib.Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR.

Use of Vaccinia Virus Smallpox Vaccine in Laboratory and Health Care Personnel at Risk for Occupational Exposure to Orthopoxviruses - Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2015.

On June 25, 2015, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with live smallpox (vaccinia) vaccine (ACAM2000) for laboratory personnel who directly handle 1) cultures or 2) animals contaminated or infected with replication-competent vaccinia virus, recombinant vaccinia viruses derived from replication-competent vaccinia strains (i.e., those that are capable of causing clinical infection and producing infectious virus in humans), or other orthopoxviruses that infect humans (e.g., monkeypox, cowpox, and variola) (recommendation category: A, evidence type 2 [Box]). Health care personnel (e.g., physicians and nurses) who currently treat or anticipate treating patients with vaccinia virus infections and whose contact with replication-competent vaccinia viruses is limited to contaminated materials (e.g., dressings) and persons administering ACAM2000 smallpox vaccine who adhere to appropriate infection prevention measures can be offered vaccination with ACAM2000 (recommendation category: B, evidence type 2 [Box]). These revised recommendations update the previous ACIP recommendations for nonemergency use of vaccinia virus smallpox vaccine for laboratory and health care personnel at risk for occupational exposure to orthopoxviruses (1). Since 2001, when the previous ACIP recommendations were developed, ACAM2000 has replaced Dryvax as the only smallpox vaccine licensed by the U.S. Food and Drug Administration (FDA) and available for use in the United States (2). These recommendations contain information on ACAM2000 and its use in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses.

Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC.

ID: 78: Recombinant MVA generating excess early double-stranded RNA transiently activates PKR and triggers enhanced innate immune responses

Poxviruses produce significant amounts of double-stranded (ds)RNA late in infection due to convergent transcription of late genes, but have evolved to avoid producing dsRNA early in infection. Cellular protein kinase R (PKR) is activated by dsRNA and can trigger important antiviral defense mechanisms including protein synthesis shutdown, apoptosis and type I interferon (IFN) secretion. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway in poxvirus-infected cells. We found that highly replication-restricted modified vaccinia virus Ankara (MVA) that was engineered to produce excess amounts of dsRNA either from EGFP gene inserts or from the endogenous E3L gene very early in infection showed enhanced induction of IFN- β in murine and human cells, despite a functional E3L gene. Increased IFN- β production was largely RIG-I-like receptor-independent, but strongly PKR-dependent and was associated with increased IRF3 activation. Excess early dsRNA-producing MVAs activated PKR very early but transiently and replicated normally in chicken embryo fibroblasts (CEFs). In mice, MVA-dsRNA mutants induced enhanced systemic levels of IFN- α , IFN- β and multiple chemokines, which was also largely depending on functional PKR. Replication-competent chorioallantois vaccinia virus Ankara (CVA) that was similarly engineered to generate excess early dsRNA was completely apathogenic in mice even at very high doses. It also induced increased amounts of IFN- β in infected cells. Thus, genetically adjuvanting MVA and CVA by generating excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to strongly attenuate vaccinia virus in vivo.

HIV-1 vaccines based on replication-competent Tiantan vaccinia protected Chinese rhesus macaques from simian HIV infection.

To assess the efficacy of HIV vaccines constructed from replication-competent Tiantan vaccinia virus (rTV) alone or combined with DNA in protecting Chinese rhesus macaques from homologous Simian/Human Immunodeficiency Virus (SHIV)-CN97001 challenge. The nef, gag, pol, and gp140 genes from strain CRF07_BC HIV-1 CN54 were selected to construct an HIV vaccine using the rTV or rTV/DNA vaccine. After vaccination, the vaccine and control groups were intravenously challenged with SHIV-CN97001 (32 MID50). HIV-specific antibodies and neutralizing antibodies, gp70 V1V2 binding antibodies, and cytotoxic T-lymphocyte responses were measured prospectively after vaccination with an ELISA, a virus infectivity assay in TZM-bl cells, and ELISPOT assays, respectively. Viral RNA was quantified after challenge with real-time reverse transcriptase-PCR (RT-PCR), and protection efficacy was determined with an analysis of CD8 lymphocyte depletion in vivo. Both rTV and DNA/rTV vaccine groups developed strong cellular and humoral responses against HIV-1 CN54 antigens, including Gag and Env, and also developed significant and persistent anti-Env antibodies and neutralizing antibodies after immunization. Both the rTV and DNA/rTV groups were significantly protected against SHIV-CN97001 or displayed lower viremia than the controls. After CD8 lymphocyte depletion, no viremia was detectable in the vaccinated monkeys, but rebounded rapidly in the control animals. Protection against infection correlated with vaccine-elicited neutralizing antibodies specific for homologous HIV-1 viruses. An rTV-based HIV-1 vaccine, with or without a DNA primer, provided protection from SHIV challenge in a macaque model. Replication-competent Tiantan vaccinia is a promising vector and should enable advances in HIV-1 vaccine development.

Study of the immunogenicity and virulence of replication-competent recombinant vaccinia virus in CD4＋T cell-depleted mice

Objective To investigate the effects of CD4＋T cell depletion in BALB/c mice on the immunogenicity and virulence of replication-competent recombinant vaccinia virus. Methods Twelve BALB/c mice were inoculated with recombinant Tiantan vaccinia （ rTV, n=8 ） or vaccinia virus Tiantan strain （ VTT, n=4） by tail scarification after the depletion of CD4＋T cells with anti-CD4 monoclonal anti-body（ McAb） injected intraperitoneally for three days before virus inoculation.A control group without anti-body treatment was set up accordingly.Several parameters including the body weight, pocks on tail, viral shedding and the percentage of CD4＋T cells were monitored.In the fourth week after virus infection, ovaries were collected from mice and viral loads in the tissue were titrated by plaque forming assay on chick embryo fibroblast （ CEF） cells.The specific cellular immune responses against vaccinia virus and HIV induced by inoculation of VTT and rTV respectively were detected by intracellular cytokine staining （ ICS） assay.ELISA was used to detect antibodies against vaccinia virus and HIV-1 gp120.Results All mice with or without CD4 McAb treatment showed typical poxvirus pocks on tails after inoculation of vaccinia viruses, but none of them developed secondary or satellite lesions.It took a longer time for CD4＋T cell-depleted mice to heal from lesions, to regain body weights and to release viruses than the mice in control group.No vaccinia virus was detected in the ovaries of CD4＋T cell-depleted mice or mice in control group.The mean absorbance（ A） values for the detection of HIV-specific and vaccinia virus-specific antibodies in CD4＋T cell-depleted mice with ELISA were respectively 0.119 and 0.168, which were significantly lower than those in mice of control group.The titers of neutralizing antibodies against vaccinia virus in McAb/rTV treated mice （1 ∶321） were lower than those in rTV treated mice （1 ∶1286） （P〈0.05）.The percentages of CD4＋T cells secreting IFN-γ（0.654%） in McAb/rTV treated mice were significantly lower than those in rTV treated mice in the fourth week after immunization （P 〈0.0004）.No significant differences with the vaccinia virus-specificCD8＋ T cell responses were observed among mice with or without CD4＋T cells depletion.Conclusion Thereplication and dissemination of replication-competent recombinant vaccinia virus could be effectively controlledin the mice with CD4＋ T cell-depletion.The depletion of CD4＋ T cells significantly reduced the humoraland CD4＋ T cell responses, but had no effect on CD8＋ T cell responses. Key words: Replication-competent recombinant vaccinia virus CD4＋T cell CD8＋T cell Viral clearance Immunogenicity

Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.

Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-β in murine and human cells in the presence of an intact E3L gene. IFN-β induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.

Vaccinia virus GLV‐1h153 in combination with 131 I shows increased efficiency in treating triple‐negative breast cancer

We investigated the therapeutic efficacy of a replication-competent oncolytic vaccinia virus, GLV-1h153, carrying human sodium iodide symporter (hNIS), in combination with radioiodine in an orthotopic triple-negative breast cancer (TNBC) murine model. In vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. Orthotopic xenografts (MDA-MB-231 cells) received intratumoral injection of GLV-1h153 or PBS. One week after viral injection, xenografts were randomized into 4 treatment groups: GLV-1h153 alone, GLV-1h153 and (131)I (∼ 5 mCi), (131)I alone, or PBS, and followed for tumor growth. Kruskal-Wallis and Wilcoxon tests were performed for statistical analysis. Radiouptake assay showed a 178-fold increase of radioiodine uptake in hNIS-expressing infected cells compared with PBS control. Systemic (131)I-iodide in combination with GLV-1h153 resulted in a 6-fold increase in tumor regression (24 compared to 146 mm(3) for the virus-only treatment group; P<0.05; d 40). We demonstrated that a novel vaccinia virus, GLV-1h153, expresses hNIS, increases the expression of the symporter in TNBC cells, and serves both as a gene marker for noninvasive imaging of virus and as a vehicle for targeted radionuclide therapy with (131)I.

Immunogenicity and safety of the vaccinia virus LC16m8Δ vector expressing SIV Gag under a strong or moderate promoter in a recombinant BCG prime-recombinant vaccinia virus boost protocol

We compared the effect of the very strong pSFJ1-10 and moderately strong p7.5 promoters on the immunogenicity and pathogenicity of the replication-competent vaccinia virus (VV) LC16m8Δ (m8Δ) vector harboring the SIV gag gene in a vaccination regimen consisting of a recombinant BCG-SIVGag (rBCG-SIVGag) prime followed by a recombinant vaccinia boost. m8Δ/pSFJ/SIVGag synthesized more Gag protein than m8Δ/p7.5/SIVGag but replicated less efficiently in vitro. In addition, m8Δ/pSFJ/SIVGag was less pathogenic and elicited Gag-specific IFN-γ+, CD107a+, CD8+ cells more efficiently than m8Δ/p7.5/SIVGag. Vaccination by this regimen elicited long-lasting Gag-specific CD8+ T cells, the majority of which showed a CCR7− phenotype at over 8 weeks post-boost. Tetramer staining analyses revealed maintenance of Gag specific tetramer+, CD62L−, CD8+ T cells for long time in vaccinated mice. However, Gag expression increased the neurotoxicity of the vaccinia vector, indicating the necessity of safety testing for each recombinant VV. We propose that this recombinant BCG prime-m8Δ/pSFJ/HIVGag boost regimen would be a promising vaccination procedure for preventing HIV infection.

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.