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.     

Development of smallpox vaccine candidates with integrated interleukin-15 that demonstrate superior immunogenicity, efficacy, and safety in mice

The potential use of variola virus, the etiological agent of smallpox, as a bioterror agent has heightened the interest in the reinitiation of smallpox vaccination. However, the currently licensed Dryvax vaccine, despite its documented efficacy in eradicating smallpox, is not optimal for the vaccination of contemporary populations with large numbers of individuals with immunodeficiencies because of severe adverse effects that can occur in such individuals. Therefore, the development of safer smallpox vaccines that can match the immunogenicity and efficacy of Dryvax for the vaccination of contemporary populations remains a priority. Using the Wyeth strain of vaccinia virus derived from the Dryvax vaccine, we generated a recombinant Wyeth interleukin-15 (IL-15) with integrated IL-15, a cytokine with potent immunostimulatory functions. The integration of IL-15 into the Wyeth strain resulted in a >1,000-fold reduction in lethality of vaccinated athymic nude mice and induced severalfold-higher cellular and humoral immune responses in wild-type mice that persisted longer than those induced by the parental Wyeth strain. The superior efficacy of Wyeth IL-15 was further demonstrated by the ability of vaccinated mice to fully survive a lethal intranasal challenge of virulent vaccinia virus even 10 months after vaccination, whereas all mice vaccinated with parental Wyeth strain succumbed. By integrating IL-15 into modified vaccinia virus Ankara (MVA), a virus currently under consideration as a substitute for the Dryvax vaccine, we developed a second vaccine candidate (MVA IL-15) with greater immunogenicity and efficacy than Dryvax. Thus, Wyeth IL-15 and MVA IL-15 viruses hold promise as more-efficacious and safe alternatives to the Dryvax vaccine.     

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.     

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).     

Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized with Escherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge.     

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.     

Application of Bioluminescence Imaging to the Prediction of Lethality in Vaccinia Virus-Infected Mice

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC = 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.In 1980, the World Health Organization declared that the world was finally free of smallpox as an extant human disease, and routine smallpox vaccination was discontinued. However, concerns that variola virus (the causative agent of smallpox) might be reintroduced into the human population as a bioterrorist agent has intensified research targeted toward the development of novel antiviral therapies and safe and effective vaccines (3, 14). The global eradication program was accomplished using several vaccines based on attenuated replicating vaccinia virus strains, including Dryvax—a vaccine based on live vaccinia virus derived from the New York City Board of Health strain (NYCBOH) and prepared from calf lymph (Wyeth Laboratories) that was used in the Unites States and West Africa (22). For a long time, Dryvax was the only U.S. licensed vaccine for human use against the smallpox virus and was considered the “gold standard” due to the long-lasting virus-neutralizing antibodies it generated (1, 13). However, due to substantial risks of developing adverse reactions, such as progressive vaccinia, eczema vaccinatum, and severe generalized vaccinia, vaccination is contraindicated in people with compromised immune systems and individuals with eczema. In addition, the Dryvax vaccine was also shown to induce transient myopericarditis in a small proportion of healthy recipients, and therefore, it is no longer recommended for general use (2). The smallpox ACAM2000 vaccine utilizing the same NYCBOH strain of vaccinia virus and propagated in African green monkey cells (Vero cells) is manufactured by Acambis/Baxter Pharmaceuticals and was recently licensed by the Food and Drug Administration for clinical use. A total of six clinical trials (phases I to III) with ACAM2000 vaccine were performed, and the results showed that vaccine-emergent reactions were slightly reduced in the ACAM2000 recipients (11). However, concerns related to stability, inability to be diluted, and decreased numbers of takes in ACAM2000 vaccine-experienced subjects suggested that further developments are needed to ensure the maintenance of a potent and safe vaccine stockpile (11). Several alternative vaccine modalities that are expected to have better safety profiles are under development or in clinical trials, such as modified vaccinia Ankara (5), DNA plasmids, and recombinant proteins. It is conceivable that new immunotherapies are needed to ensure safe and efficient treatment of complications associated with live vaccinia virus-based vaccines and for treatment of subjects exposed to variola virus.Intravenous vaccinia virus immunoglobulin (VIGIV; Cangene Corporation) is a new product that replaced intramuscular vaccinia virus immunoglobulin (VIGIM), which had been used for treatment of complications related to smallpox vaccinations since the 1950s. VIGIV is prepared from the purified gamma immunoglobulin (immunoglobulin G) fraction of plasma taken from healthy donors previously vaccinated with Dryvax who demonstrate high titers of vaccinia virus neutralizing antibody. This product was licensed for the treatment of patients who develop complications following Dryvax vaccination. It has been suggested that VIGIV might also be life saving for unvaccinated persons who have come into contact with people exposed to variola virus itself and may help to limit the spread of the disease (27). The efficacy of VIGIV has not been tested in many clinical situations. The clinical pharmacology and pharmacodynamics of VIGIV were tested in two phase I clinical trials, which showed that the product was well tolerated, with all adverse effects related to VIGIV being typical of those expected following intravenous administration of the protein (27). The recommended dosage of VIGIV for the management of several complications of smallpox (vaccinia) vaccinations is 6,000 U/kg body weight (4).The development of novel vaccines against smallpox and of new pre- or postexposure therapies critically depends on the use of animal models for the initial preclinical testing. Several animal models are employed as surrogate models of variola virus infection in humans, including infection of macaques with monkeypox virus, which has been used as a tool to dissect the immune responses to poxviruses (21). Importantly, experiments with rhesus macaques demonstrated a critical role for antibodies in the protection of vaccinated animals and helped select an optimal vaccine regimen utilizing DNA-coded monkeypox virus proteins in proof-of-concept studies (6, 15). However, due to the expense and the requirement for biosafety level 3 facilities, nonhuman primate models cannot be widely used for smallpox vaccine development. The majority of preclinical testing and initial characterization of smallpox vaccines and therapies is performed with the Western Reserve (WR) strain of vaccinia virus or with ectomelia virus, which is highly lethal in mice. Various endpoints are used to follow infections in mice, including weight loss, pox lesion scoring, and viral-load measurements by plaque formation on sensitive cell lines. These endpoints are not optimal, as they cannot avoid morbidity or accurately predict lethality in individual animals. They require very large number of animals in order to determine survival rates and to quantify viral loads in internal organs. Lethality was frequently used as an endpoint in the past but is no longer acceptable.Whole-animal bioimaging has been widely used for studies of microbial and viral pathogens in small-animal models (16, 19). This technology allows monitoring of pathogen dissemination in real time, locating pathogens residing in unexpected anatomical sites, and greatly reducing the numbers of animals required per study by providing spatial and temporal information for individual animals (16, 19). For this purpose, genes coding for luciferase enzymes are expressed in bacterial or viral pathogens, and dissemination of the recombinant pathogen is recorded by detecting light emitted from the tissues of infected live animals (19). Using bioimaging of live animals, bioluminescence of poxviruses expressing luciferase was employed to evaluate mucosal vaccinia virus vaccine (7), to dissect the roles of type I interferons and innate immunity in controlling viral replication and spread (18), and to characterize the tissue distribution of and immune responses induced by viral strains used for the development of vectored vaccines against other pathogens (9, 23). Measurements of photon fluxes have been shown to correlate in linear fashion with viral loads in internal organs in vaccinia virus-infected mice, suggesting that bioluminescence provides a direct measure of viral dissemination (19). However, whether bioluminescence signals derived from luciferase-expressing vaccinia virus can be used to predict lethality in infected mice has not yet been investigated.To that end, we used a recombinant WR vaccinia virus expressing luciferase to record bioluminescence in healthy mice after intranasal (i.n.) infection with 1 to 5 50% lethal doses (LD₅₀). Groups of animals were immunized with Dryvax or pretreated with VIGIV. We used acquired images from surviving and nonsurviving mice to calculate total fluxes in internal organs and applied receiver operating characteristic (ROC) curve analysis to generate predictive models. Our data showed that measurements of total fluxes in the spleen and liver 3 and 5 days postinfection provided strong predictive models of lethality. The predictive power of bioimaging was further investigated when bioluminescences from several organs and multiple time points were combined in multiple logistic regression analysis.     

Modified vaccinia virus Ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin-4

Recent events have raised concern over the use of pathogens, including variola virus, as biological weapons. Vaccination with Dryvax is associated with serious side effects and is contraindicated for many people, and the development of a safer effective smallpox vaccine is necessary. We evaluated an attenuated vaccinia virus, modified vaccinia virus Ankara (MVA), by use of a murine model to determine its efficacy against an intradermal (i.d.) or intranasal (i.n.) challenge with vaccinia virus (vSC8) or a recombinant vaccinia virus expressing murine interleukin-4 that exhibits enhanced virulence (vSC8-mIL4). After an i.d. challenge, 15 of 16 mice who were inoculated with phosphate-buffered saline developed lesions, one dose of intramuscularly administered MVA was partially protective (3 of 16 mice developed lesions), and the administration of two or three doses of MVA was completely protective (0 of 16 mice developed lesions). In unimmunized mice, an i.n. challenge with vSC8 caused a significant but self-limited illness, while vSC8-mIL4 resulted in lethal infections. Immunization with one or two doses of MVA prevented illness and reduced virus titers in mice who were challenged with either vSC8 or vSC8-mIL4. MVA induced a dose-related neutralizing antibody and vaccinia virus-specific CD8+-T-cell response. Mice immunized with MVA were fully protected from a low-dose vSC8-mIL4 challenge despite a depletion of CD4+ cells, CD8+ cells, or both T-cell subsets or an antibody deficiency. CD4+- or CD8+-T-cell depletion reduced the protection against a high-dose vSC8-mIL4 challenge, and the depletion of both T-cell subsets was associated with severe illness and higher vaccinia virus titers. Thus, MVA induces broad humoral and cellular immune responses that can independently protect against a molecularly modified lethal poxvirus challenge in mice. These data support the continued development of MVA as an alternative candidate vaccine for smallpox.     

Subunit recombinant vaccine protects against monkeypox

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.     

Genomic Sequence and Virulence of Clonal Isolates of Vaccinia Virus Tiantan,the Chinese Smallpox Vaccine Strain

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.     

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.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.     

HLA alleles associated with the adaptive immune response to smallpox vaccine: a replication study

We previously reported HLA allelic associations with vaccinia virus (VACV)-induced adaptive immune responses in a cohort of healthy individuals (n = 1,071 subjects) after a single dose of the licensed smallpox (Dryvax) vaccine. This study demonstrated that specific HLA alleles were significantly associated with VACV-induced neutralizing antibody (NA) titers (HLA-B*13:02, *38:02, *44:03, *48:01, and HLA-DQB1*03:02, *06:04) and cytokine (HLA-DRB1*01:03, *03:01, *10:01, *13:01, *15:01) immune responses. We undertook an independent study of 1,053 healthy individuals and examined associations between HLA alleles and measures of adaptive immunity after a single dose of Dryvax-derived ACAM2000 vaccine to evaluate previously discovered HLA allelic associations from the Dryvax study and determine if these associations are replicated with ACAM2000. Females had significantly higher NA titers than male subjects in both study cohorts [median ID50 discovery cohort 159 (93, 256) vs. 125 (75, 186), p < 0.001; replication cohort 144 (82, 204) vs. 110 (61, 189), p = 0.024]. The association between the DQB1*03:02 allele (median ID₅₀ discovery cohort 152, p = 0.015; replication cohort 134, p = 0.010) and higher NA titers was replicated. Two HLA associations of comparable magnitudes were consistently found between DRB1*04:03 and DRB1*08:01 alleles and IFN-γ ELISPOT responses. The association between the DRB1*15:01 allele with IFN-γ secretion was also replicated (median pg/mL discovery cohort 182, p = 0.052; replication cohort 203, p = 0.014). Our results suggest that smallpox vaccine-induced adaptive immune responses are significantly influenced by HLA gene polymorphisms. These data provide information for functional studies and design of novel candidate smallpox vaccines.     

Development of Eczema Vaccinatum in Atopic Mouse Models and Efficacy of MVA Vaccination against Lethal Poxviral Infection

Smallpox vaccine based on live, replicating vaccinia virus (VACV) is associated with several potentially serious and deadly complications. Consequently, a new generation of vaccine based on non-replicating Modified vaccinia virus Ankara (MVA) has been under clinical development. MVA seems to induce good immune responses in blood tests, but it is impossible to test its efficacy in vivo in human. One of the serious complications of the replicating vaccine is eczema vaccinatum (EV) occurring in individuals with atopic dermatitis (AD), thus excluding them from all preventive vaccination schemes. In this study, we first characterized and compared development of eczema vaccinatum in different mouse strains. Nc/Nga, Balb/c and C57Bl/6J mice were epicutaneously sensitized with ovalbumin (OVA) or saline control to induce signs of atopic dermatitis and subsequently trans-dermally (t.d.) immunized with VACV strain Western Reserve (WR). Large primary lesions occurred in both mock- and OVA-sensitized Nc/Nga mice, while they remained small in Balb/c and C57Bl/6J mice. Satellite lesions developed in both mock- and OVA-sensitized Nc/Nga and in OVA-sensitized Balb/c mice with the rate 40–50%. Presence of mastocytes and eosinophils was the highest in Nc/Nga mice. Consequently, we have chosen Nc/Nga mice as a model of AD/EV and tested efficacy of MVA and Dryvax vaccinations against a lethal intra-nasal (i.n.) challenge with WR, the surrogate of smallpox. Inoculation of MVA intra-muscularly (i.m.) or t.d. resulted in no lesions, while inoculation of Dryvax t.d. yielded large primary and many satellite lesions similar to WR. Eighty three and 92% of mice vaccinated with a single dose of MVA i.m. or t.d., respectively, survived a lethal i.n. challenge with WR without any serious illness, while all Dryvax-vaccinated animals survived. This is the first formal prove of protective immunity against a lethal poxvirus challenge induced by vaccination with MVA in an atopic organism.     

Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human beta-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human alpha-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 x 10(5) PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10-50 micro M), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.     

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.     

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.     

Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.     

Identification of protective T-cell antigens for smallpox vaccines

Background aimsE3L is an immediate-early protein of vaccinia virus (VV) that is detected within 0.5 h of infection, potentially before the many immune evasion genes of vaccinia can exert their protective effects. E3L is highly conserved among orthopoxviruses and hence could provide important protective T-cell epitopes that should be retained in any subunit or attenuated vaccine. We have therefore evaluated the immunogenicity of E3L in healthy VV-vaccinated donors.MethodsPeripheral blood mononuclear cells from healthy volunteers (n = 13) who had previously received a smallpox vaccine (Dryvax) were activated and expanded using overlapping E3L peptides and their function, specificity and antiviral activity was analyzed. E3L-specific T cells were expanded from 7 of 12 (58.3%) vaccinated healthy donors. Twenty-five percent of these produced CD8+ T-cell responses and 87.5% produced CD4+ T cells. We identified epitopes restricted by HLA-B35 and HLA-DR15.ResultsE3L-specific T cells killed peptide-loaded target cells as well as vaccinia-infected cells, but only CD8+ T cells could prevent the spread of infectious virus in virus inhibition assays. The epitopes recognized by E3L-specific T cells were shared with monkeypox, and although there was a single amino acid change in the variola epitope homolog, it was recognized by vaccinia-specific T-cells.ConclusionsIt might be important to include E3L in any deletion mutant or subunit vaccine and E3L could provide a useful antigen to monitor protective immunity in humans.