DNA Vaccines - A Modern Gimmick or a Boon to Vaccinology?

The reports in 1993 that naked DNA encoding viral genes conferred protective immunity came as a surprise to most vaccinologists. This review analyses the expanding number of examples where plasmid DNA induces immune responses. Issues such as the type of immunity induced, mechanisms of immune protection, and how DNA vaccines compare with other approaches are emphasized. Additional issues discussed include the likely means by which DNA vaccines induce CTL, how the potency and type of immunity induced can be modified, and whether DNA vaccines represent a practical means of manipulating unwanted immune response occurring during immunoinflammatory diseases. It seems doubtful if DNA vaccines will replace currently effective vaccines, but they may prove useful for prophylactic use against some agents that at present lack an effective vaccine. DNA vaccines promise to be valuable to manipulate the immune response in situations where responses to agents are inappropriate or ineffective.

Poxvirus viability and signatures in historical relics.

Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it.

Novel poxvirus in big brown bats, northwestern United States.

A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of Poxviridae.

Phylogenetic and ecologic perspectives of a monkeypox outbreak, southern Sudan, 2005.

Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans.

Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246, and CMX001.

Progressive vaccinia (PV) is a rare but potentially lethal complication that develops in smallpox vaccine recipients with severely impaired cellular immunity. We describe a patient with PV who required treatment with vaccinia immune globulin and who received 2 investigational agents, ST-246 and CMX001. We describe the various molecular, pharmacokinetic, and immunologic studies that provided guidance to escalate and then successfully discontinue therapy. Despite development of resistance to ST-246 during treatment, the patient had resolution of PV. This case demonstrates the need for continued development of novel anti-orthopoxvirus pharmaceuticals and the importance of both intensive and timely clinical and laboratory support in management of PV.

Design and Optimization of a Monkeypox virus Specific Serological Assay.

Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak.

Detection of North American orthopoxviruses by real time-PCR.

The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad.

Human monkeypox outbreak caused by novel virus belonging to Congo Basin clade, Sudan, 2005.

To determine the outbreak source of monkeypox virus (MPXV) infections in Unity State, Sudan, in November 2005, we conducted a retrospective investigation. MPXV was identified in a sub-Sahelian savannah environment. Three case notification categories were used: suspected, probable, and confirmed. Molecular, virologic, and serologic assays were used to test blood specimens, vesicular swabs, and crust specimens obtained from symptomatic and recovering persons. Ten laboratory-confirmed cases and 9 probable cases of MPXV were reported during September-December 2005; no deaths occurred. Human-to-human transmission up to 5 generations was described. Our investigation could not fully determine the source of the outbreak. Preliminary data indicate that the MPXV strain isolated during this outbreak was a novel virus belonging to the Congo Basin clade. Our results indicate that MPXV should be considered endemic to the wetland areas of Unity State. This finding will enhance understanding of the ecologic niche for this virus.

Magnitude and diversity of immune response to vaccinia virus is dependent on route of administration.

Historic observations suggest that survivors of smallpox maintained lifelong immunity and protection to subsequent infection compared to vaccinated individuals. Although protective immunity by vaccination using a related virus (vaccinia virus (VACV) strains) was the key for smallpox eradication, it does not uniformly provide long term, or lifelong protective immunity (Heiner et al., 1971). To determine differences in humoral immune responses, mice were inoculated with VACV either systemically, using intranasal inoculation (IN), or locally by an intradermal (ID) route. We hypothesized that sub-lethal IN infections may mimic systemic or naturally occurring infection and lead to an immunodominance reaction, in contrast to localized ID immunization. The results demonstrated systemic immunization through an IN route led to enhanced adaptive immunity to VACV-expressed protein targets both in magnitude and in diversity when compared to an ID route using a VACV protein microarray. In addition, cytokine responses, assessed using a Luminex® mouse cytokine multiplex kit, following IN infection was greater than that stemming from ID infection. Overall, the results suggest that the route of immunization (or infection) influences antibody responses. The greater magnitude and diversity of response in systemic infection provides indirect evidence for anecdotal observations made during the smallpox era that survivors maintain lifelong protection. These findings also suggest that systemic or disseminated host immune induction may result in a superior response, that may influence the magnitude of, as well as duration of protective responses.

IMVAMUNE® and ACAM2000® Provide Different Protection against Disease When Administered Postexposure in an Intranasal Monkeypox Challenge Prairie Dog Model.

The protection provided by smallpox vaccines when used after exposure to Orthopoxviruses is poorly understood. Postexposu re administration of 1st generation smallpox vaccines was effective during eradication. However, historical epidemiological reports and animal studies on postexposure vaccination are difficult to extrapolate to today's populations, and 2nd and 3rd generation vaccines, developed after eradication, have not been widely tested in postexposure vaccination scenarios. In addition to concerns about preparedness for a potential malevolent reintroduction of variola virus, humans are becoming increasingly exposed to naturally occurring zoonotic orthopoxviruses and, following these exposures, disease severity is worse in individuals who never received smallpox vaccination. This study investigated whether postexposure vaccination of prairie dogs with 2nd and 3rd generation smallpox vaccines was protective against monkeypox disease in four exposure scenarios. We infected animals with monkeypox virus at doses of 104 pfu (2× LD50) or 106 pfu (170× LD50) and vaccinated the animals with IMVAMUNE® or ACAM2000® either 1 or 3 days after challenge. Our results indicated that postexposure vaccination protected the animals to some degree from the 2× LD50, but not the 170× LD5 challenge. In the 2× LD50 challenge, we also observed that administration of vaccine at 1 day was more effective than administration at 3 days postexposure for IMVAMUNE®, but ACAM2000® was similarly effective at either postexposure vaccination time-point. The effects of postexposure vaccination and correlations with survival of total and neutralizing antibody responses, protein targets, take formation, weight loss, rash burden, and viral DNA are also presented.

Encephalitis after secondary smallpox vaccination.

We describe a case of post-secondary vaccination encephalitis in a smallpox vaccine recipient and discuss detection of intrathecal antibody to vaccinia virus as a potential diagnostic test.

In vitro efficacy of ST246 against smallpox and monkeypox.

Since the eradication of smallpox and the cessation of routine childhood vaccination for smallpox, the proportion of the world's population susceptible to infection with orthopoxviruses, such as variola virus (the causative agent of smallpox) and monkeypox virus, has grown substantially. In the United States, the only vaccines for smallpox licensed by the Food and Drug Administration (FDA) have been live virus vaccines. Unfortunately, a substantial number of people cannot receive live virus vaccines due to contraindications. Furthermore, no antiviral drugs have been fully approved by the FDA for the prevention or treatment of orthopoxvirus infection. Here, we show the inhibitory effect of one new antiviral compound, ST-246, on the in vitro growth properties of six variola virus strains and seven monkeypox virus strains. We performed multiple assays to monitor the cytopathic effect and to evaluate the reduction of viral progeny production and release in the presence of the compound. ST-246 had 50% effective concentrations of

A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein.

Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines.

Monkeypox zoonotic associations: insights from laboratory evaluation of animals associated with the multi-state US outbreak.

At the onset of the 2003 US monkeypox outbreak, virologic data were unavailable regarding which animal species were involved with virus importation and/or subsequent transmission to humans and whether there was a risk for establishment of zoonotic monkeypox in North America. Similarly, it was unclear which specimens would be best for virus testing. Monkeypox DNA was detected in at least 33 animals, and virus was cultured from 22. Virus-positive animals included three African species associated with the importation event (giant pouched rats, Cricetomys spp.; rope squirrels, Funisciuris sp.; and dormice, Graphiuris sp.). Virologic evidence from North American prairie dogs (Cynomys sp.) was concordant with their suspected roles as vectors for human monkeypox. Multiple tissues were found suitable for DNA detection and/or virus isolation. These data extend the potential host range for monkeypox virus infection and supports concern regarding the potential for establishment in novel reservoir species and ecosystems.

Assessing Monkeypox Virus Prevalence in Small Mammals at the Human-Animal Interface in the Democratic Republic of the Congo.

During 2012, 2013 and 2015, we collected small mammals within 25 km of the town of Boende in Tshuapa Province, the Democratic Republic of the Congo. The prevalence of monkeypox virus (MPXV) in this area is unknown; however, cases of human infection were previously confirmed near these collection sites. Samples were collected from 353 mammals (rodents, shrews, pangolins, elephant shrews, a potamogale, and a hyrax). Some rodents and shrews were captured from houses where human monkeypox cases have recently been identified, but most were trapped in forests and agricultural areas near villages. Real-time PCR and ELISA were used to assess evidence of MPXV infection and other Orthopoxvirus (OPXV) infections in these small mammals. Seven (2.0%) of these animal samples were found to be anti-orthopoxvirus immunoglobulin G (IgG) antibody positive (six rodents: two Funisciurus spp.; one Graphiurus lorraineus; one Cricetomys emini; one Heliosciurus sp.; one Oenomys hypoxanthus, and one elephant shrew Petrodromus tetradactylus); no individuals were found positive in PCR-based assays. These results suggest that a variety of animals can be infected with OPXVs, and that epidemiology studies and educational campaigns should focus on animals that people are regularly contacting, including larger rodents used as protein sources.

Evaluation of human-to-human transmission of monkeypox from infected patients to health care workers.

BACKGROUND: In 2003, human monkeypox was first identified in the United States. The outbreak was associated with exposure to infected prairie dogs, but the potential for person-to-person transmission was a concern. This study examines health care worker (HCW) exposure to 3 patients with confirmed monkeypox. METHODS: Exposed HCWs, defined as HCWs who entered a 2-m radius surrounding case patients with confirmed monkeypox, were identified by infection-control practitioners. A self-administered questionnaire and analysis of paired serum specimens determined exposure status, immune response, and postexposure signs and symptoms of monkeypox. RESULTS: Of 81 exposed HCWs, 57 (70%) participated in the study. Among 57 participants, 40 (70%) had > or =1 unprotected exposure; none reported signs or symptoms consistent with monkeypox illness. One exposed HCW (2%), who had been vaccinated for smallpox within the past year, had serological evidence of recent orthopoxvirus infection; acute- and convalescent-phase serum specimens tested positive for anti-orthopoxvirus IgM. No exposed HCWs had signs and symptoms consistent with monkeypox. CONCLUSION: More than three-quarters of exposed HCWs reported at least 1 unprotected encounter with a patient who had monkeypox. One asymptomatic HCW showed laboratory evidence of recent orthopoxvirus infection, which was possibly attributable to either recent infection or smallpox vaccination. Transmission of monkeypox likely is a rare event in the health care setting.

Transmission of monkeypox among persons exposed to infected prairie dogs in Indiana in 2003.

OBJECTIVE: To describe a cluster of human monkeypox cases associated with exposure to ill prairie dogs in a home child care. DESIGN, SETTING, PARTICIPANTS: We identified all persons exposed to 2 pet prairie dogs in County A, Indiana; performed active surveillance for symptomatic monkeypox infection; and evaluated the types of exposure that may have resulted in infection. For children who attended the child care where the animals were housed, we also measured the rate of seroconversion to monkeypox virus. MAIN OUTCOME MEASURES: Nine (13%) of 70 persons exposed to the prairie dogs reported signs and symptoms of monkeypox. Two (40%) of 5 symptomatic child care attendees reported direct contact with the prairie dogs. Two (13%) of 15 child care attendees evaluated tested positive for IgM antibodies against orthopoxvirus; both reported symptoms consistent with monkeypox. RESULTS: The risk of symptomatic infection correlated with the time and intensity of animal exposure, which was 100% (4/4) among family members with extensive direct contact, 19% (5/26) among the veterinarian and nonfamily child care attendees with moderate exposure, and 0% (0/40) among school children with limited exposure (P<.01). CONCLUSIONS: Monkeypox virus was transmitted from ill prairie dogs in a child care and veterinary facilities. The risk of symptomatic infection correlated with the amount of exposure to the prairie dogs. Although most cases of human monkeypox were associated with direct animal contact, other routes of transmission cannot be excluded.

A phylogeographic investigation of African monkeypox.

Monkeypox is a zoonotic disease caused by a virus member of the genus Orthopoxvirus and is endemic to Central and Western African countries. Previous work has identified two geographically disjuct clades of monkeypox virus based on the analysis of a few genomes coupled with epidemiological and clinical analyses; however, environmental and geographic causes of this differentiation have not been explored. Here, we expand previous phylogenetic studies by analyzing a larger set of monkeypox virus genomes originating throughout Sub-Saharan Africa to identify possible biogeographic barriers associated with genetic differentiation; and projected ecological niche models onto environmental conditions at three periods in the past to explore the potential role of climate oscillations in the evolution of the two primary clades. Analyses supported the separation of the Congo Basin and West Africa clades; the Congo Basin clade shows much shorter branches, which likely indicate a more recent diversification of isolates within this clade. The area between the Sanaga and Cross Rivers divides the two clades and the Dahomey Gap seems to have also served as a barrier within the West African clade. Contraction of areas with suitable environments for monkeypox virus during the Last Glacial Maximum, suggests that the Congo Basin clade of monkeypox virus experienced a severe bottleneck and has since expanded its geographic range.

Laboratory Investigations of African Pouched Rats (Cricetomys gambianus) as a Potential Reservoir Host Species for Monkeypox Virus.

Monkeypox is a zoonotic disease endemic to central and western Africa, where it is a major public health concern. Although Monkeypox virus (MPXV) and monkeypox disease in humans have been well characterized, little is known about its natural history, or its maintenance in animal populations of sylvatic reservoir(s). In 2003, several species of rodents imported from Ghana were involved in a monkeypox outbreak in the United States with individuals of three African rodent genera (Cricetomys, Graphiurus, Funisciurus) shown to be infected with MPXV. Here, we examine the course of MPXV infection in Cricetomys gambianus (pouched Gambian rats) and this rodent species' competence as a host for the virus. We obtained ten Gambian rats from an introduced colony in Grassy Key, Florida and infected eight of these via scarification with a challenge dose of 4X104 plaque forming units (pfu) from either of the two primary clades of MPXV: Congo Basin (C-MPXV: n = 4) or West African (W-MPXV: n = 4); an additional 2 animals served as PBS controls. Viral shedding and the effect of infection on activity and physiological aspects of the animals were measured. MPXV challenged animals had significantly higher core body temperatures, reduced activity and increased weight loss than PBS controls. Viable virus was found in samples taken from animals in both experimental groups (C-MPXV and W-MPXV) between 3 and 27 days post infection (p.i.) (up to 1X108 pfu/ml), with viral DNA found until day 56 p.i. The results from this work show that Cricetomys gambianus (and by inference, probably the closely related species, Cricetomys emini) can be infected with MPXV and shed viable virus particles; thus suggesting that these animals may be involved in the maintenance of MPXV in wildlife mammalian populations. More research is needed to elucidate the epidemiology of MPXV and the role of Gambian rats and other species.