Tenth anniversary of smallpox eradication: Results of epidemiological and virological surveillance and studies in the post-eradication period

In accordance with recommendations of the Global Commission on the certification of smallpox eradication for the 10-year period after the eradication of this infection, all suspected cases of smallpox have been thoroughly checked up, and in none of them the diagnosis of smallpox has been confirmed. The study of monkeypox in humans has revealed that this zoonosis is spread over a wider area than supposed earlier and covers 7 countries of Equatorial Africa, occurring most frequently in Zaire. In about 70% of cases of monkeypox in human the disease is contracted from animals serving as natural virus carriers and in about one-third of such cases, from humans having monkeypox. The infectivity of humans with monkeypox for persons having close contacts with them is somewhat lower (12.3%) than in smallpox when this characteristic varies from 37% to 88%. Monkeypox in humans may take an asymptomatic course. Some species of tropical squirrels serve as natural virus carriers. These investigations have also resulted in essential corrections being made in understanding the ecology of cowpox virus, another orthopoxvirus pathogenic for man. At least 5 species of rodents have proved to be of interest as natural carriers of cowpox virus.     

Study of allergic reactions in the body after experimental smallpox vaccination

Experiments on guinea pigs demonstrated a mixed cellular-humoral character of allergy in smallpox vaccination. The ratio of cellular and humoral components depended on the site of the vaccina application. Skin manifestations in smallpox vaccination (hyperemia, infiltration, papula) were due to the multiplication of the virus proper, increased sensitivity of the surrounding tissues to the live vibrio, and specific immunological reconstruction. The skin of the sensitized animal after the anaphylactic shock in response to the intracardiac injection of the challenging dose of the vaccine virus acquired areactivity to its subsequent application.     

Lack of correlation between complications after smallpox vaccination and the ABO blood-group system

Blood group [ABO system] distribution was analysed in 245 children with complications after smallpox vaccination. One hundred and thirty-two children had neurological and 113 and skin or mucosal complications. 41.6% of the children were blood-group A or AB. 58.4% were blood group O or B, which was not a significant difference from the normal population [44.7%---48.7% and 51.0%---55.9%, respectively]. The analysis did not show in persons possessing blood groups A or AB an enhanced susceptibility to complications following antismallpox immunization or to particularly severe clinical forms of these complications. Immunological advantages in persons with blood group O or B were not found either.     

Characterization of UVC light sensitivity of vaccinia virus

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m(2), three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.     

Characterization of UVC Light Sensitivity of Vaccinia Virus

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m², three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.     

Isolation of smallpox virus from the nasopharynx of patients with variola sine eruptione

Summary In 2 out of 13 contacts of smallpox patients, variola virus was recovered from nasopharyngeal secretions on the 14th and the 18th day after the last contact. One of them was vaccinated, and showed transient prodromal symptoms, the other, who had never beee vaccinated did not show signs of illness. Evidence has been obtained from this investigation, that a few cases of smallpox during a local epidemic, which could not be explained by contact with a smallpox patient, may be explained by contact with a virus carrier.     

Clinical studies of vaccine TEOVac under the conditions of remote revaccination

In the clinical trials of vaccine TEOVac in lower and reglamented doses under the conditions of remote vaccination, carried out on 11 volunteers, local reactions in the form of hyperemia, gingival edema (1 vaccinee), faucial hyperemia, enlargement of submaxillary lymph nodes (2 vaccinees) were registered in some of the vaccines; in one vaccinee systemic postvaccinal reaction of medium gravity was observed. Revaccination produced no negative effect on the hematological and biochemical characteristics of the blood, as well as on the urine characteristics. The study of the sensitizing vaccine to the antigens of the accumulation substrate (chick embryo), as well as its influence on the development of autoimmune reactions, revealed the absence of the allergic action of the preparation and its influence on autoimmune processes in the vaccines. The trial of the smallpox vaccine in tablets under the conditions of the primary immunization of adults was the topical and most promising trend in the improvement of smallpox vaccination, as the preparation TEOVac proved to be safe, in contrast to the traditional smallpox vaccine introduced by the scarification method, for both vaccines and nonimmunized persons having contacts with them.     

Human monkey pox: its clinico-epidemiological characteristics

During the course of the smallpox eradication programme, a new eruptive disease clinically resembling smallpox was discovered in Zaire. The disease, which was named monkeypox after the virus, is a zoonosis occurring sporadically in countries of western and central Africa with tropical rain forest. The studies carried out in Zaire from 1980 through 1985 showed that monkeypox affects mainly children in relatively small remote villages whose population has traditionally frequent contacts with wild animals. Apart from the wildlife, the virus can be transmitted from man to man, but among other sources of infection sick persons did not exceed 20%. Presumed human transmission has occurred in 38 out of 61 outbreaks of human monkeypox and only once reached the third and once the fourth generation; the transmission in all affected villages under observation has extinguished itself. Considering the sporadic and relatively rare occurrence of the disease and expected complications following the immunization with vaccinia which protects from monkeypox, introduction of mass vaccination in the areas at risk is hardly justified at present.     

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.     

AB0 blood groups and smallpox in a rural population of West Bengal and Bihar (India)

The problem was reexamined whether there is a relationship between the AB0 blood groups and incidence, severity and outcome of smallpox. In persons suffering from smallpox, and living in rural areas of West Bengal and Bihar, India, severity and outcome of the disease were registered, and the AB0 blood groups were determined. Healthy siblings were used as controls for incidence. Four series were available:

1. 1.
   200 suffering patients and 200 controls examined during summer 1965 in the Burdwan district. West Bengal.     
   
   

Vaccinia virus persistence in a child against the background of immune deficiency

A young girl, vaccinated against smallpox 6 years before suffered from a persistent vaccinia virus infection and a congenital skin disease, i.e. epidermolysis bullosa. The virus was isolated from skin lesions at the vaccination site and remote sites and repeatedly from the blood; it was not isolated from bone-marrow specimen, saliva, pharynx or urine. The titre of virus-neutralizing antibodies was low (1:10), immunoglobulins A, M and G were within age-related limits; antibodies against measles and tetanus were at protective levels, skin tests were positive. Staphylococcal antitoxin titre was extremely high. The child's mother, not vaccinated against smallpox, possessed vaccinia--virus-neutralizing antibodies at high titre (over 1:320). Examination of the child did not show any quantitative immune deficiency. Immune deviations were found in the lymphocyte blast-transformation reaction on stimulation with PHA and specific antigen, as well as in the nonspecific-suppression test. The possible genesis of the virus persistence and the role of the virus in the clinical course of the disease are discussed.     

Deletion of the monkeypox virus inhibitor of complement enzymes locus impacts the adaptive immune response to monkeypox virus in a nonhuman primate model of infection

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.     

Unraveling the structure of the variola topoisomerase IB-DNA complex: a possible new twist on smallpox therapy

Smallpox is a serious and highly contagious disease that is caused by the variola virus. It is one of the most severe infectious human diseases known, with mortality rates as high as 30%. A successful worldwide vaccination program led to the eradication of smallpox in 1980. However, the high transmission rate of variola virus, coupled with the deadly nature of smallpox, makes this virus a potentially devastating weapon for bioterrorism. Currently, there is no specific treatment for smallpox. However, a recent article on the structure of a variola topoisomerase IB-DNA complex provides an intriguing starting point for the rational design of drugs with potential activity against smallpox.     

Inactivation of poxviruses by upper-room UVC light in a simulated hospital room environment

In the event of a smallpox outbreak due to bioterrorism, delays in vaccination programs may lead to significant secondary transmission. In the early phases of such an outbreak, transmission of smallpox will take place especially in locations where infected persons may congregate, such as hospital emergency rooms. Air disinfection using upper-room 254 nm (UVC) light can lower the airborne concentrations of infective viruses in the lower part of the room, and thereby control the spread of airborne infections among room occupants without exposing occupants to a significant amount of UVC. Using vaccinia virus aerosols as a surrogate for smallpox we report on the effectiveness of air disinfection, via upper-room UVC light, under simulated real world conditions including the effects of convection, mechanical mixing, temperature and relative humidity. In decay experiments, upper-room UVC fixtures used with mixing by a conventional ceiling fan produced decreases in airborne virus concentrations that would require additional ventilation of more than 87 air changes per hour. Under steady state conditions the effective air changes per hour associated with upper-room UVC ranged from 18 to 1000. The surprisingly high end of the observed range resulted from the extreme susceptibility of vaccinia virus to UVC at low relative humidity and use of 4 UVC fixtures in a small room with efficient air mixing. Increasing the number of UVC fixtures or mechanical ventilation rates resulted in greater fractional reduction in virus aerosol and UVC effectiveness was higher in winter compared to summer for each scenario tested. These data demonstrate that upper-room UVC has the potential to greatly reduce exposure to susceptible viral aerosols. The greater survival at baseline and greater UVC susceptibility of vaccinia under winter conditions suggest that while risk from an aerosol attack with smallpox would be greatest in winter, protective measures using UVC may also be most efficient at this time. These data may also be relevant to influenza, which also has improved aerosol survival at low RH and somewhat similar sensitivity to UVC.     

A rapid detection method for Vaccinia virus, the surrogate for smallpox virus

Prior to the World Health Organization's announcement of total eradication in 1977 [J. Am. Med. Assoc. 281 (1999) 1735], smallpox was a worldwide pathogen. Vaccinations were ceased in 1980 and now with a largely unprotected world population, smallpox is considered the ideal biowarfare agent [Antiviral Res. 57 (2002) 1]. Infection normally occurs after implantation of the virus on the oropharyngeal or respiratory mucosa [J. Am. Med. Assoc. 281 (1999) 2127]. Smallpox virus can be detected from the throats of exposed individuals prior to onset of illness and prior to the infectious stage of the illness. A rapid, sensitive real-time assay to detect Variola virus (smallpox) has been developed using the Vaccinia virus, a surrogate of smallpox, as a target. Cyanine 5 dye-labeled anti-Vaccinia antibody was used in a sandwich immunoassay to produce a fluorescent signal in the presence of the Vaccinia virus. The signal was detected using the Analyte 2000 biosensor (Research International, Monroe, WA). The Analyte 2000 uses a 635 nm laser diode to provide excitation light that is launched into a polystyrene optical waveguide. Fluorescent molecules within the evanescent wave are excited and a portion of their emission energy recouples into the waveguide. A photodiode quantifies the emission light at wavelengths between 670 and 710 nm. The biosensor was able to detect a minimum of 2.5 x 10(5) pfu/ml of Vaccinia virus in seeded throat culture swab specimens.     

Comparative study of the smallpox vaccines from B-51, EM-63 and L-IVP in a controlled epidemiological experiment. II. The characteristiics of the immunogenicity of the smallpox vaccines]

Immunogenicity of smallpox vaccines prepared of EM-63, L-IVP, and B-51 strains was studied under conditions of strict controlled epidemiological trial. Skin reactions to revaccination and vaccines antigenic activity indices were detemined in the persons vaccinated. Changes in the virus-neutralizing and antibodies suppressing hemagglutination was the same in persons vaccinated with any of the preparations tested. The maximal virus-neutralizing antibodies level was determined 1 month after the vaccination and persisted without any essential changes for one year. The titre of hemagglutination inhibiting antibodies also reached the maximum in one month, but diminished gradually by the end of one year after the vaccination. There were found no significant differences in the antigenic activity of the vaccines. The vaccines studied also displayed no difference in the number and character of skin reactions to revaccination. In comparing the antibodies level and the character of skin reactions to revaccination it was found that the titres of hemagglutination inhibiting antibodies and virus-neutralizing antibodies of 1:40 and over were in the great majority of cases determined in the blood sera of the vaccinated persons with the immediate and negative reactions to revaccination, i. e. in those with intensive postvaccinal immunity.     

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.     

The threat of smallpox

Smallpox is a highly contagious disease mainly transmitted by aerosols with a high case-fatality. The smallpox virus has evolved from a long adaptation to humans during Evolution, explaining that the virus is highly specific for humans and nonpathogenic for animals. Smallpox was eradicated in 1977 and vaccination was abandoned in the 1980's. This virus is a dreadful potential biological weapon since the reemergence of smallpox on the planet might be expected to be devastating, due to its high 'contagiosity', which would rapidly spread in naive populations, especially those living in urban areas, and worldwide through air travels. There is no anti-viral treatment and vaccine is active in the first four days post-exposure. Today, the stocks of smallpox virus constitute one of the most dangerous threats for humanity. There is a need for improving the safety of the vaccine and to reconsider the preventive strategy to face a possible attack by smallpox virus.     

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.