Smallpox Infection Research Articles

Antiviral Immunity following Smallpox Virus Infection: a Case-Control Study

Outbreaks of smallpox (i.e., caused by variola virus) resulted in up to 30% mortality, but those who survived smallpox infection were regarded as immune for life. Early studies described the levels of neutralizing antibodies induced after infection, but smallpox was eradicated before contemporary methods for quantifying T-cell memory were developed. To better understand the levels and duration of immunity after smallpox infection, we performed a case-control study comparing antiviral CD4(+) and CD8(+) T-cell responses and neutralizing antibody levels of 24 smallpox survivors with the antiviral immunity observed in 60 smallpox-vaccinated (i.e., vaccinia virus-immune) control subjects. We found that the duration of immunity following smallpox infection was remarkably similar to that observed after smallpox vaccination, with antiviral T-cell responses that declined slowly over time and antiviral antibody responses that remained stable for decades after recovery from infection. These results indicate that severe, potentially life-threatening disease is not required for the development of sustainable long-term immunity. This study shows that the levels of immunity induced following smallpox vaccination are comparable in magnitude to that achieved through natural variola virus infection, and this may explain the notable success of vaccination in eradicating smallpox, one of the world's most lethal diseases.

Vitiligo iridis in patients with a history of smallpox infection

Vitiligo iridis in patients with a history of smallpox infection

Methods to measure vaccine immunity

The biggest triumph in vaccination is the eradication of smallpox. Ironically, no methods to measure vaccine immunity were set in place. Protection and immunity was achieved without detailed analysis nor knowledge of cellular or humoral immunity induction. Today, owing to strict health and safety issues of vaccine manufacturing, vaccines must meet higher standards of safety and biochemical characterization than they did in the past. This has led to the production of highly purified vaccines, and the identification and isolation of the antigens responsible for protection. A vaccine cannot move forward without the demonstration of effective immunity induced by the regime. Moreover, contrary to the vaccination strategy that eradicated smallpox and other acute viral infections, several modern vaccines aim at the eradication of established chronic diseases such as chronic viral infections or cancer. As the pathogens or cancer antigens are generally intracellular, modern vaccines can rely less effectively on the neutralizing properties of antibody responses that cannot cross the cell membrane, and depend predominantly on T-cell-based recognition of affected cells for their elimination. Thus, new technologies have to be developed to measure vaccine immunity. Are we going round and round in circles? Are we making it harder for ourselves? Is vaccine development getting too hard? Why do we have to demonstrate immunity, if we can show protection? No regulatory agency will take a vaccine forward that is not highly purified and can demonstrate immunity induction. Several methods of measuring humoral and cellular immunity have been required to be developed to meet this criteria. Some methods include ELISA, cytotoxic T-lymphocyte (CTL) assay, CTL precursor frequency assay, T-cell proliferation assays, carboxyfluorescein diacetate succinimidyl ester assays, intracellular and extracellular cytokine production by cells in culture using either ELISA or multiplex and flow cytometry, polyfunctional T-cell assays, ELISpot and MHC class I/II tetramers. Thus, there is an enormous amount of information and reagents available for guiding vaccine and immunotherapeutics development. This special issue on ‘Methods to measure vaccine immunity’ focuses on a number of recent and promising approaches used to measure immunity induced following vaccination. Topics included in this special focus issue are as follows: Gene-expression profiling in vaccine therapy and immunotherapy for cancer [1]. The use of microarrays is described in order to understand the mechanism by which tumors are rejected; Antibody-profiling technologies for studying humoral responses to infectious agents [2]. Protein microarrays are used to evaluate antibody responses to thousands of antigens at one time. The luciferase immunoprecipitation system overcomes some drawbacks of the conventional protein microarray assay. These new technologies offer a new tool for understanding humoral immunity induction to proteins; Vaccine-induced antibody responses in patients with carcinoma [3]. A nice overview of current methods utilized for measuring antibody responses and for assessing their antitumor efficacy in the context of clinical trials; Methods to measure T-cell responses [4]. A mini review on recent methods used to measure T-cell immunity, such as the use of CTL, CTL precursor frequency assays, carboxyfluorescein diacetate succinimidyl ester, flow cytometric approaches, spectrophotometric approaches and use of ovalbumin-specific CD8+ T cells from OTI and OTII T-cell receptor tansgenic mice; New flow cytometric assays for monitoring cell-mediated cytotoxicity [5]. A comparison of IFN-γ ELISpot, granzyme B ELISpot and conventional 51Cr-release assays is given. A detailed analysis of the use of flow cytometric methods to measure T-cell immunity induction is demonstrated; Evaluation of cellular immune responses in cancer vaccine recipients [6]. Measurement of cellular immunity is addressed using NY-ESO-1 as an example and techniques such as delayed-type hypersensitivity, measurement of T cells from peripheral blood, use of overlapping peptides to measure CD4 and CD8 T cells, and the role of Treg cells in vaccine immunity induction are described; Development and application of phosphoflow as a tool for immunomonitoring [7]. This article describes the new flow cytometry technology, phosphoflow for measuring multiple intracellular signaling molecules in the immune system at a single-cell level for lymphocyte immune monitoring; Surface plasmon resonance for vaccine design and efficacy studies [8]. An outline of how surface plasmon resonance biosensors are emerging to understand how serum is used to monitor antibody immunity; The challenges of assessing infant vaccine responses in resource-poor settings [9]. A very nice and comprehensive article on methods to measure vaccine immune responses where only poor settings are available. There is a vast amount of information already available to promote a greater understanding into the role of specific immune induction to a vaccination regime. The use of newer immune monitoring tools as described in this special focus issue will serve to improve immune monitoring and determine the effectiveness of vaccine strategies for the treatment or immunotherapeutic approaches to diseases.

A novel highly reproducible and lethal nonhuman primate model for orthopox virus infection.

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1–3 days after onset of symptoms, even when very low infectious viral doses of 5×102 pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID50 (minimal monkey infectious dose 50%) of 8.3×102 pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.

Smallpox: An eradicated infection with persistent sequels – Case report and a brief on smallpox

Sequelae of smallpox infection on the ocular surface are still seen, including corneal scars adherent leukoma and phthisical globes. This paper will report another sequel of smallpox infection causing inadvertent bleb in a 62-year-old diabetic female with no history of ocular surgery or trauma in either eye. The patient had smallpox infection during her childhood. Her follow up extended from May 1997 until August 2007 with a constant eye examinations including controlled intraocular pressure, avascular cystic inadvertent bleb, and up drown peaked pupil.

Smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships

Smallpox (infection with Orthopoxvirus variola) remains a feared illness more than 25 years after its eradication. Historically, case-fatality rates (CFRs) varied between outbreaks (<1 to approximately 40 %), the reasons for which are incompletely understood. The extracellular enveloped virus (EEV) form of orthopoxvirus progeny is hypothesized to disseminate infection. Investigations with the closely related Orthopoxvirus vaccinia have associated increased comet formation (EEV production) with increased mouse mortality (pathogenicity). Other vaccinia virus genetic manipulations which affect EEV production inconsistently support this association. However, antisera against vaccinia virus envelope protect mice from lethal challenge, further supporting a critical role for EEV in pathogenicity. Here, we show that the increased comet formation phenotypes of a diverse collection of variola viruses associate with strain phylogeny and geographical origin, but not with increased outbreak-related CFRs; within clades, there may be an association of plaque size with CFR. The mechanisms for variola virus pathogenicity probably involves multiple host and pathogen factors.

ST-246 Antiviral Efficacy in a Nonhuman Primate Monkeypox Model: Determination of the Minimal Effective Dose and Human Dose Justification

Therapeutics for the treatment of pathogenic orthopoxvirus infections are being sought. In the absence of patients with disease, animal models of orthopoxvirus disease are essential for evaluation of the efficacies of antiviral drugs and establishment of the appropriate dose and duration of human therapy. Infection of nonhuman primates (NHP) by the intravenous injection of monkeypox virus has been used to evaluate a promising therapeutic drug candidate, ST-246. ST-246 administered at 3 days postinfection (which corresponds to the secondary viremia stage of disease) at four different doses (from 100 mg/kg of body weight down to 3 mg/kg) once a day for 14 days was able to offer NHP 100% protection from a lethal infection with monkeypox virus and reduce the viral load and lesion formation. In NHP, the administration of ST-246 at a dose of 10 mg/kg/day for 14 days resulted in levels of blood exposure comparable to the levels attained in humans administered 400 mg in the fed state. These results suggest that administration of an oral dosage of 400 mg once daily for 14 days will be effective for the prevention or treatment of smallpox or monkeypox infections in humans.

Immunity from Smallpox Vaccine Persists for Decades: A Longitudinal Study

Purpose The threat of smallpox resulting from bioterrorist action has prompted a reassessment of the level of immunity in current populations. Methods We have examined the magnitude and duration of antiviral antibody immunity conferred by smallpox vaccination in 246 participants of the Baltimore Longitudinal Study of Aging. Of this population, 209 subjects were vaccinated one or more times 13 to 88 years before this evaluation, and stored serum samples were available at various intervals after vaccination. An additional 8 subjects who had documented childhood smallpox infection and 29 subjects with no history of infection or vaccination were included. We quantified the total vaccinia IgG and neutralizing antibody titers in each of these subgroups of participants over time. Results Vaccinated participants maintained antivaccinia IgG and neutralizing antibody titers above 3 natural logs essentially indefinitely. The absolute titer of antivaccinia antibody was only slightly higher after multiple vaccinations. In 97% of the participants, no decrease in vaccinia-specific antibody titers was noted with age over a follow-up period of up to 88 years. Moreover, Baltimore Longitudinal Study of Aging participants who survived active smallpox infections in their youth retained antivaccinia antibody titers that were similar to the levels detected in vaccinated subjects. Conclusion These data suggest that multiple or recent vaccinations are not essential to maintain vaccinia-specific antibody responses in human subjects. Scarce vaccine supplies should be applied first to individuals who have not previously been vaccinated.

Evaluation of orally delivered ST-246 as postexposure prophylactic and antiviral therapeutic in an aerosolized rabbitpox rabbit model

Orthopoxviruses, such as variola and monkeypox viruses, can cause severe disease in humans when delivered by the aerosol route, and thus represent significant threats to both military and civilian populations. Currently, there are no antiviral therapies approved by the U.S. Food and Drug Administration (FDA) to treat smallpox or monkeypox infection. In this study, we showed that administration of the antiviral compound ST-246 to rabbits by oral gavage, once daily for 14 days beginning 1 h postexposure (p.e.), resulted in 100% survival in a lethal aerosolized rabbitpox model used as a surrogate for smallpox. Furthermore, efficacy of delayed treatment with ST-246 was evaluated by beginning treatment on days 1, 2, 3, and 4 p.e. Although a limited number of rabbits showed less severe signs of the rabbitpox disease from the day 1 and day 2 p.e. treatment groups, their illness resolved very quickly, and the survival rates for these group of rabbits were 88% and 100%, respectively. But when the treatment was started on days 3 or 4 p.e., survival was 67% and 33%, respectively. This work suggests that ST-246 is a very potent antiviral compound against aerosolized rabbitpox in rabbits and should be investigated for further development for all orthopoxvirus diseases.

Poxvirus-Based Vaccine Platforms: Getting at Those Hard-To-Reach Places

Rodolfo Nino-Fong & James B Johnston† †Author for correspondence Institute for Nutrisciences & Health, National Research Council Canada, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada Tel.: +1 902 566 8007; Fax: +1 902 367 7539; james.johnston@nrc.ca ‘The prominence of poxviruses as candidate vaccine vectors completes a circle begun in the 18th century when Jenner first tested the theory of vaccination using Cowpox virus to inoculate against smallpox infection.’

Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens

The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

Real time PCR for the assessment of CD8+ T cellular immune response after prophylactic vaccinia vaccination

Background The magnitude of specific CD8+ T cell reactivity responsible for vaccine-induced protection against smallpox infection has not yet been fully elucidated. Among other techniques, RT-PCR for the monitoring of cytokine release in effector T cells against tumor and viral antigens has demonstrated a novel promising method. Objective To determine the functional status of antigen specific CD8+ T cells in healthy participants before and 4 weeks after prophylactic vaccination (Lister strain) against smallpox using quantitative real-time PCR (qRT-PCR). Study design Changes of interferon-γ (IFNγ) mRNA expression levels on short term ex vivo peptide antigen stimulation were measured. The corresponding specific CD8+ T cell reactivity was then displayed as CD8-normalized IFN-γ levels (IFN-γ/CD8 ratio). Results We found a 5–9 fold increase of CD8+ T cell reactivity in three out of four vaccinated individuals. The kinetics and strength determined in responders reveal a virus specific T cell effector repertoire pre-vaccination and a corresponding functional state after immunization comparable also to data obtained from tetramer- and ELISPOT analysis. Conclusions Apart from protective vaccinia-specific neutralizing antibodies, the presence of antigen-specific CD8+ T-cells has been demonstrated after vaccinia vaccination. In concordance with others, results from this PCR-based study indicate that this smallpox vaccine induces strong vaccinia virus-specific CD8+ and IFN-γ producing T cell responses.

Study of the role of CCR5 in a mouse model of intranasal challenge with Yersinia pestis

CCR5 is a chemokine receptor used by HIV-1 to enter cells and has recently been found to act as a pathogen associated molecule pattern receptor. Current positive selection for the high frequency of a CCR5-Δ32 allele in humans has been attributed to resistance to HIV, smallpox, and plague infections. Using an intranasal mouse model of Y. pestis infection, we have found that lack of CCR5 does not enhance host resistance to Y. pestis infection and that CCR5-mediated responses might have a protective role. CCR5−/− mice exhibited higher levels of circulating RANTES and MIP-1α than those exhibited by wild-type mice at the baseline and throughout the course of Y. pestis infection. High levels of RANTES and MIP-1α, which are CCR5 ligands that mediate Natural Killer cell migration, may reflect compensation for the absence of CCR5 signaling.

Proteome‐wide analysis of the serological response to vaccinia and smallpox

The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax, and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A-type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross-reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola-infected individuals and Dryvax vaccinees were indistinguishable. This is the first description of antibody-specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.

Dramatic rescue relieves rare case of smallpox infection

Dramatic rescue relieves rare case of smallpox infection

Intranasal cowpox virus infection of the mouse as a model for preclinical evaluation of smallpox vaccines

The intranasal infection of mice with cowpox virus (CPXV) has been evaluated as a model for smallpox infection in man. Administration of a lethal dose of CPXV allowed time for development of T-cell responses but antibodies could not be detected before death occurred. In contrast, infection with a sublethal dose was associated with an early T-cell response followed by neutralising antibodies which correlated with virus clearance. Comparison of two first generation smallpox vaccines revealed no significant differences in terms of immunogenicity, protection and post-challenge virus clearance. These studies show that the CPXV/mouse model is valuable for the initial assessment of smallpox vaccines.

Real-Time PCR to Identify Variola Virus or Other Human Pathogenic Orthopox Viruses

Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism. We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L. The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses. This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.

Can We Eradicate Measles?

In 1977, health officials in Somalia documented the last case of naturally acquired smallpox, completing an intensive, wide-ranging campaign to eradicate the global incidence of smallpox infections. Several features made this feat possible, including that humans are the only known hosts (and reservoir) for this virus, that it spreads to susceptible individuals only when it is producing a rash in an infected host, that recovery from smallpox provides lifelong immunity, and that the vaccine provides long-term protection from infection (see table).

Discovery of Monkeypox in Sudan

To the Editor: On November 4, 2005, the Poxvirus Program at the Centers for Disease Control and Prevention (CDC), a World Health Organization (WHO) Collaborating Center for Smallpox and other Poxvirus Infections, was contacted by a former volunteer with Medecins sans Frontieres (MSF) with a request for information regarding a child with a generalized pustular rash illness in southern Sudan. On the basis of a review of a photograph of the child (Figure 1), dated October 28, 2005, and limited clinical information describing a febrile period of disease, it was agreed that the findings were suggestive of a systemic orthopoxvirus . . .

Identification of Vaccinia CD8 + T-Cell Epitopes Conserved among Vaccinia and Variola Viruses Restricted by Common MHC Class I Molecules, HLA-A2 or HLA-B7

Immunization with vaccinia virus results in long-lasting protection against smallpox and is an approach that has been successfully used to eliminate natural smallpox infections worldwide. Today, vaccinia virus is very important not only as a vaccine virus to protect human against smallpox, but also as an expression vector for immunization against other infectious diseases, such as HIV and cancer. In this article, we identify three new vaccinia human CD8 + T-cell epitopes conserved among vaccinia and variola viruses restricted by HLA-A2, HLA-B7, or HLA-B*3502, which belongs to the HLA-B7 supertype. Identification of these CD8 + T-cell epitopes restricted by common HLA alleles will help to quantitate human CD8 + T-cell responses to licensed and experimental smallpox vaccines and to vaccinia virus vectors. CD8 + T-cell responses specific to these epitopes can also be used to quantitate cellular immune responses, especially with new smallpox vaccines that do not induce a “take,” such as the modified vaccinia virus Ankara strain. Combined with previous reports by us and others, these results show that there are some vaccinia viral proteins containing multiple epitopes restricted by different MHC molecules of humans and mice.