[EVALUATION OF THE HUMAN SENSITIVITY TO SMALLPOX VIRUS BY THE PRIMARY CULTURES OF THE MONOCYTE-MACROPHAGES].

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.

Vaccinia virus induces rapid necrosis in keratinocytes by a STAT3-dependent mechanism.

RATIONALE: Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. METHODS: To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. RESULTS: Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. CONCLUSIONS: Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.

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.

[Interferon inductor cupping of postvaccinal complications after variolation].

Efficacy of arbidol and ridostin in cupping postvaccinal complications due to variolation was studied by the clinico-virological, hematological and biochemical indices and it was shown that arbidol was efficient in cupping development of dermal complications, lowered the severity of the postvaccinal reaction and stimulated the cellular and humoral immune response. Ridostin, a high molecular interferon inductor, was highly efficient in cupping all the forms of the postvaccinal complications, including the neurological and cutaneous ones.

Effect of vaccination with modified vaccinia Ankara (ACAM3000) on subsequent challenge with Dryvax.

BACKGROUND: Despite the success of smallpox vaccination, the immunological correlates of protection are not fully understood. To investigate this question, we examined the effect of immunization with modified vaccinia Ankara (MVA) on subsequent challenge with replication-competent vaccinia virus (Dryvax). METHODS: Dryvax challenge by scarification was conducted in 36 healthy subjects who had received MVA (n = 29) or placebo (n = 7) in a previous study of doses and routes of immunization. Subjects were followed up for clinical take, viral shedding, and immune responses. RESULTS: MVA administration attenuated clinical takes in 21 (72%) of 29 subjects, compared with 0 of 7 placebo recipients (P = .001). Attenuation was most significant in MVA groups that received 1 x 10(7) median tissue culture infective doses (TCID(50)) intradermally (P = .001) and 1 x 10(7) TCID(50) intramuscularly (P = .001). Both duration and peak titer of viral shedding were reduced in MVA recipients. Peak neutralizing antibody responses to vaccinia virus or MVA previously induced by MVA immunization were associated with attenuated takes (P = .02) and reduced duration (P = .001) and titer (P = .005) of viral shedding. CONCLUSIONS: MVA immunization results in clinical and virologic protection against Dryvax challenge. Protection is associated with prior induction of neutralizing antibodies to MVA or vaccinia virus. MVA administered intradermally has protective and immunologic responses similar to those of a 10-fold-higher dose given subcutaneously.

Kinetics of serum cytokines after primary or repeat vaccination with the smallpox vaccine.

BACKGROUND: The smallpox vaccine is associated with more serious adverse events than any other live attenuated vaccine in use today. Although studies have examined serum cytokine levels in primary vaccine recipients at 1 and 3-5 weeks after vaccination with the smallpox vaccine, serial measurements have not been performed, and studies in revaccinated subjects have not been conducted. METHODS: We analyzed cytokine responses in both primary vaccine recipients and revaccinated subjects every other day for 2 weeks after vaccination. RESULTS: Primary vaccine recipients had maximal levels of granulocyte-colony-stimulating factor on days 6-7 after vaccination; peak levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptor 1, interferon (IFN)-gamma, IFN-inducible protein-10 (IP-10), interleukin (IL)-6, and tissue inhibitor of metalloproteinases-1 on days 8-9 after vaccination; peak levels of soluble TNF receptor 2 and monokine induced by IFN-gamma (MIG) on days 10-11 after vaccination; and peak levels of granulocyte-macrophage-colony-stimulating factor on days 12-13 after vaccination. Primary vaccine recipients were significantly more likely to have higher peak levels of IFN-gamma, IP-10, and MIG after vaccination than were revaccinated subjects. Primary vaccine recipients were significantly more likely to have fatigue, lymphadenopathy, and headache, as well as a longer duration of these symptoms and more hours missed from work, compared with revaccinated subjects. CONCLUSIONS: The increased frequency and duration of symptoms observed in primary vaccine recipients, compared with revaccinated subjects, paralleled the increases in serum cytokine levels in these individuals. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00325975.

Expansion, reexpansion, and recall-like expansion of Vgamma2Vdelta2 T cells in smallpox vaccination and monkeypox virus infection.

Little is known about the in vivo kinetics of T-cell responses in smallpox/monkeypox. We showed that macaque Vgamma2Vdelta2 T cells underwent 3-week-long expansion after smallpox vaccine immunization and displayed simple reexpansion in association with sterile anti-monkeypox virus (anti-MPV) immunity after MPV challenge. Virus-activated Vgamma2Vdelta2 T cells exhibited gamma interferon-producing effector function after phosphoantigen stimulation. Surprisingly, like alphabeta T cells, suboptimally primed Vgamma2Vdelta2 T cells in vaccinia virus/cidofovir-covaccinated macaques mounted major recall-like expansion after MPV challenge. Finally, Vgamma2Vdelta2 T cells localized in inflamed lung tissues for potential regulation. Our studies provide the first in vivo evidence that viruses, despite their inability to produce exogenous phosphoantigen, can induce expansion, reexpansion, and recall-like expansion of Vgamma2Vdelta2 T cells and stimulate their antimicrobial cytokine response.

Comparison of the safety and immunogenicity of ACAM1000, ACAM2000 and Dryvax in healthy vaccinia-naive adults.

Currently, more than half of the world's population has no immunity against smallpox variola major virus. This phase I double-blind, randomized trial was conducted to compare the safety and immunogenicity of two clonally derived, cell-culture manufactured vaccinia strains, ACAM1000 and ACAM2000, to the parent vaccine, Dryvax. Thirty vaccinia-naïve subjects were enrolled into each of three groups and vaccines were administered percutaneously using a bifurcated needle at a dose of 1.0x10(8)PFU/mL. All subjects had a primary skin reaction indicating a successful vaccination. The adverse events, 4-fold neutralizing antibody rise and T cell immune responses were similar between the groups.

Pharmacodynamics of cidofovir for vaccinia virus infection in an in vitro hollow-fiber infection model system.

Variola major virus remains a potent weapon of bioterror. There is currently an investigational-new-drug application for cidofovir for the therapy of variola major virus infections. Stittelaar and colleagues compared the levels of effectiveness of postexposure smallpox vaccination (Elstree-RIVM) and antiviral treatment with cidofovir or an acyclic nucleoside phosphonate analogue 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) after lethal intratracheal infection of cynomolgus monkeys with monkeypox virus, a variola virus surrogate. Their results demonstrated that either compound was more effective than vaccination with the Ellstree vaccine (K. J. Stittelaar et al., Nature 439:745-748, 2006). An unanswered question is how to translate this information into therapy for poxvirus infections in people. In a proof-of-principle study, we used a novel in vitro hollow-fiber infection model system to determine the pharmacodynamics of vaccinia virus infection of HeLa-S3 cells treated with cidofovir. Our results demonstrate that the currently licensed dose of cidofovir of 5 mg/kg of body weight weekly with probenecid (which ameliorates nephrotoxicity) is unlikely to provide protection for patients intentionally exposed to Variola major virus. We further demonstrate that the antiviral effect is independent of the schedule of drug administration. Exposures (area under the concentration-time curve) to cidofovir that will have a robust protective effect will require doses that are 5 to 10 times that currently administered to humans. Such doses may cause nephrotoxicity, and therefore, approaches that include probenecid administration as well as schedules of administration that will help ameliorate the uptake of cidofovir into renal tubular epithelial cells need to be considered when addressing such treatment for people.

Protection induced in mice against a lethal orthopox virus by the Lister strain of vaccinia virus and modified vaccinia virus Ankara (MVA).

The Lister (Elstree) strain of vaccinia virus, used in Israel for vaccination against smallpox, was studied in tissue cultures and in a mouse model. The virus failed to reach the brain of the mice when inoculated intranasally at a dose of 500,000 plaque forming units, but was lethal for 50% of them, when injected intracranially. Lower doses of virus injected intracranially caused some weight loss initially, but later the mice completely recovered. Modified vaccinia virus Ankara (MVA), when infected intranasally, did not spread beyond the lungs to other organs of the mice. Even when the mice were inoculated with MVA intracranially, they were not affected. Significant protection against a lethal dose of an orthopoxvirus was obtained in mice following immunization with the Lister strain, while larger doses and repeated vaccination procedure, were required with MVA. The Lister virus stock applied in Israel, was found to be heterogeneous in its plaque morphology. Two variants isolated from it, showed significant attenuation for mice, when inoculated intranasally and intracranially, as compared to a third variant and to the unpurified stock of the virus.

Validity of self-assessment of skin reaction after smallpox vaccination.

OBJECTIVE: Smallpox vaccinees should be evaluated for the presence of a major skin reaction ("take") one week after vaccination, but this could prove to be logistically infeasible in the context of an emergency mass-vaccination campaign. We validated a tool for self-evaluation of the vaccination site for presence of take. METHODS: We conducted a prospective, double-blinded, paired-measurement validation study of 174 non-naive adult vaccinees and their physician evaluators. Subjects provided paired, blinded, independent assessments of take 7-9 days after vaccination. RESULTS: Overall, vaccinees and evaluators agreed on 157 of 174 (90.2%) take assessments. Sensitivity of the tool was 99.1%, and specificity was 75%. The positive predictive value of self-assessment was 87.2% and the negative predictive value was 98%. Specificity of the tool and measures of agreement were significantly modified by age, education, and occupation. When adjusted for the expected take rate among a population including naive vaccinees, positive predictive value and overall agreement increased significantly. CONCLUSIONS: Self-assessment may be a feasible option for evaluation of take in the event of mass smallpox vaccination. The predictive values and overall agreement of the tool are satisfactory, and can be expected to increase when used in a largely naive population.

An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection.

The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 10(7) PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.

The nonreplicating smallpox candidate vaccines defective vaccinia Lister (dVV-L) and modified vaccinia Ankara (MVA) elicit robust long-term protection.

Current smallpox vaccines are live vaccinia viruses that replicate in the vaccinee inducing immunity against the deadly disease smallpox. Replication resulting in virus spread within the host, however, is the major cause of severe postvaccinal adverse events. Therefore, attenuated strains such as modified vaccinia Ankara (MVA) or LC16m8 are candidates as next generation vaccines. These strains are usually grown in primary cells in which mass production is difficult and have an unknown protective potential in humans. Proven vaccine strains of defined origin and modern production techniques are therefore desirable. In this study, defective vaccinia virus (dVV) lacking a gene essential for replication (derived from the Lister vaccine in a complementing cell line) was compared with the Wyeth smallpox vaccine strain and with MVA in mouse animal models using cowpox and ectromelia virus challenge. Similar to MVA, prime-boost immunizations with defective vaccinia induced robust long-term immunity, suggesting it as a promising next generation smallpox vaccine.

Differential regulation of granzyme and perforin in effector and memory T cells following smallpox immunization.

Primary immunization of healthy adults with vaccinia virus induces a local vesicle or "take" in the majority of vaccinees that previously has been shown to correlate with protection against smallpox. However, the immunologic mechanisms underlying this protective response in humans are not well characterized. We have studied human CD8+ T cells for the expression patterns of phenotypic markers and cytolytic effector molecules before and after primary smallpox immunization using nine-color polychromatic flow cytometry. One month after immunization, vaccinees developed vaccinia virus-specific CD8+ T cells with an effector cell phenotype containing both granzyme A and granzyme B. One year after immunization, we found a significant decrease in granzyme B containing cells and an increased memory cell phenotype in virus-specific CD8+ T cells. Perforin was rarely expressed directly ex vivo, but was highly expressed after Ag-specific activation in vitro. Together, these data suggest an important role for effector CD8+ T cells in controlling poxvirus infection, and have implications for our understanding of human CD8+ T cell differentiation.

Design and development of oral drugs for the prophylaxis and treatment of smallpox infection.

Smallpox was eradicated by the World Health Organization (WHO) vaccination campaign in the 1970s and the variola virus was restricted to repositories in the United States and Russia. Recently, however, concerns have arisen about the possible existence of variola outside these sites and the potential for using the virus as a weapon of bioterror. The world population now has little residual immunity to smallpox and supplies of the smallpox vaccine are being reconstituted. Large numbers of individuals with various skin diseases or immunosuppression owing to AIDS or organ transplantation medications, or who are pregnant or have heart disease might not be ideal candidates for vaccination with the current live vaccines. It would be useful to have an orally active drug that could be self-administered in case of an outbreak of smallpox.

Long-lived poxvirus immunity, robust CD4 help, and better persistence of CD4 than CD8 T cells.

The currently used smallpox vaccine is associated with a high incidence of adverse events, and there is a serious need for a safe and effective alternative vaccine. Here, we carried out a longitudinal evaluation of vaccinia virus-specific CD4 and CD8 T cells in smallpox-vaccinated individuals by using a highly sensitive intracellular cytokine staining assay. Our results demonstrate that, in addition to the CD8 response, the smallpox vaccinations raised a robust CD4 response with a Th1-dominant cytokine profile. These CD4 T cells were stable and exhibited only a twofold contraction between peak effector and memory phases compared with an approximate sevenfold contraction for CD8 cells. A significant proportion of vaccinated individuals lost detectable CD8 memory while maintaining CD4 memory. After a booster immunization, these individuals generated a robust CD8 response, which some of them rapidly lost. Thus, the current smallpox vaccine provides long-lasting CD4 help that may be critical for long-lived B-cell memory. We suggest that the provision of adequate CD4 help for CD8 and humoral effector functions will be critical to the success of the next generation of smallpox vaccines.

Clonal vaccinia virus grown in cell culture as a new smallpox vaccine.

Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination. To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line. We characterized six cloned and one uncloned vaccine candidates. One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity. By most measures, ACAM1000 was less virulent than Dryvax. We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study. The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.