Smallpox and Vaccinia

Why Smallpox Is Important

Smallpox was widespread and had a relatively high mortality rate, making it one of the most deadly of all infectious diseases. Its impact on human civilization cannot be overstated. It also represents the only human disease to be eradicated. This was made possible by the lack of an animal reservoir, the lack of a chronic carrier state, availability of a heat-stable highly efficacious vaccine, a vaccination technique that was simple and effective, intensive surveillance and containment procedures, and a united, global effort. Although it has been 44 years since the last naturally occurring case of smallpox, the disease and vaccine remain relevant due to outbreaks of zoonotic orthopoxviruses, the emergence of novel orthopoxviruses, and concerns about the use of poxviruses such as variola in bioterrorism.

Clinical Description

Smallpox is caused by either of two closely related viruses, variola major or variola minor, that can be distinguished by polymerase chain reaction (PCR) analysis. Clinically they are similar, but variola minor cases are associated with fewer systemic symptoms, less-extensive rash, little persistent scarring, and far fewer deaths. The disease has an incubation period of approximately 12 days, with a range of 7–17 days. A 2- to 3-day prodrome of high fever, malaise, and prostration with headache and backache is followed by the development of a maculopapular rash ( Fig. 55.2 ). The rash appears first on the mucosa of the mouth and pharynx, then the face and the forearms, and then spreads centrifugally to the legs and trunk. There is a slow evolution over approximately 6 or more days as the rash becomes vesicular and then pustular. The pustules are characteristically round, tense, and deeply embedded in the dermis. Crusts begin to form about the eighth or ninth day. When they separate, they leave pigment-free skin and frequently pitted scars. The eruption is characteristically more extensive on the cooler parts of the body, perhaps because the virus grows better at 35°C than 37°C. Lesions are most numerous on the face, arms, and legs ( Fig. 55.2 ) and are often found on the palms and soles. Death usually occurs late in the first week or during the second week of the illness, and although the cause of death is not definitively known, evidence suggests that it is not the result of bacterial sepsis ; rather it is probably from the effects of an overwhelming viremia, the formation of immune complexes, and the associated immunopathology, or “cytokine storm.” ^,

The WHO classification scheme for smallpox describes five types: (a) ordinary, (b) flat (also known as “malignant”), (c) hemorrhagic, (d) vaccine modified, and (e) variola sine eruptione. Rao used this classification scheme to analyze the frequency and case-fatality rates of vaccinated and unvaccinated cases in Madras, India. Among persons not previously vaccinated, the vast majority of variola major cases (approximately 89%) were of the ordinary type and had a fatality rate of approximately 30%. Flat smallpox accounted for 6.7% of cases and had an associated mortality of 96%. The hemorrhagic type accounted for 2.4% of cases and was more common in pregnant women; 96% died, with nearly 100% fetal wastage. Extensive bleeding into the skin and the gastrointestinal tract preceded death; in retrospect, hemorrhagic cases almost certainly represented disseminated intravascular coagulation. Cases “modified” by prior vaccination had many fewer lesions and, in the series by Rao, accounted for 2.1% of the cases and resulted in no deaths. The rash in such cases was usually more scant and atypical, and the evolution of lesions more rapid. Variola sine eruptione (without rash) was rare and occurred in highly immune persons who had laboratory evidence of infection but were not infectious to others.

Complications

Complications of smallpox include bacterial infections of the skin and other organs, pneumonia, generalized sepsis, destructive arthritis in young children and arthropathies, corneal ulceration resulting in blindness (most frequent in patients with trachoma), keratitis, osteomyelitis, and encephalitis. The latter, present in approximately one in 500 cases, is indistinguishable from the acute postinfectious perivascular demyelination observed as a complication of infection with vaccinia, measles, and varicella. Cases of smallpox among pregnant women usually result in spontaneous abortion or, occasionally, a stillborn infant with evidence of lesions on the skin.

The most common sequelae among survivors are facial pockmarks, blindness, and limb deformities. The scars, present in 65–80% of survivors, are usually depigmented and most evident on the face and result from the destruction of sebaceous glands followed by shrinking of granulation tissue and fibrosis.

Virology

Variola virus belongs to the genus Orthopoxvirus , family Poxviridae, which includes vaccinia, monkeypox, cowpox, camelpox, and mousepox (also known as ectromelia or ECTV). All species exhibit extensive serologic cross-reactivity in vitro and in experimental animals. The poxvirus genome, the largest of all virions that infect humans, is an ovoid brick-shaped structure with a diameter of approximately 200 nm, consisting of a single molecule of a double-stranded DNA ( Fig. 55.3 ). It differs from most other DNA viruses in that it multiplies in the cytoplasm rather than in the nucleus of susceptible cells.

The orthopoxviruses grow and produce a cytopathic effect in cultured cells derived from many species, ^, although they generally grow best in cells from humans and other primates. The four that infect humans (variola, vaccinia, cowpox, and monkeypox viruses) cannot be differentiated readily from one another in most cell cultures. For diagnostic purposes, therefore, they are customarily grown on the chorioallantoic membrane of 10- to 12-day-old chick embryos, on which they produce pocks characteristic of their species. Newer PCR techniques have been developed that permit their differentiation (see “Diagnosis” later).

Although inactivated fairly rapidly by direct sunlight, especially under hot or humid conditions, Variola virus exhibits considerable durability in lesion scabs (hundreds of days, and in one case up to 13 years) ^, or embedded in cotton (months to years). A variety of historical artifacts (Egyptian mummies from 1200–1100 BC, 17th to 19th century corpses exhumed from permafrost, corpses exhumed from crypts or coffins from the 1500s to 1800s, a museum exhibit containing a scab from a child infected in 1876) have also been tested for the presence of variola virus. Although in some cases viral particles have been identified by electron microscopy and viral DNA isolated by PCR, none of these relics has yielded infectious virus.

Pathogenesis as It Relates to Prevention

Smallpox generally follows the progression in the host described by Fenner and colleagues for ectromelia. Smallpox infection occurs by implantation of variola virus on the respiratory mucosa. The virus is usually transmitted as virions in droplets expressed from nasal and oropharyngeal secretions. Higher concentrations of virus are believed to be expelled by patients with the hemorrhagic and malignant forms of the disease. When cough is present, the virus can be expelled as a fine-particle aerosol, but cough is not a typical symptom of ordinary smallpox. After migration to and multiplication in regional lymph nodes, asymptomatic viremia develops about the third or fourth day, followed by multiplication of virus in the spleen, bone marrow, and lymph nodes. A secondary viremia occurs some 8–10 days later, accompanied by high temperature, toxemia, and prostration. The virus, contained in leukocytes, then localizes in small blood vessels of the dermis and beneath the oral and pharyngeal mucosa, where it subsequently infects adjacent cells. In the skin, this process causes the characteristic maculopapular lesions that then evolve into vesicular and pustular lesions. Lesions are more extensive on the face and extremities, perhaps because the virus grows most readily at temperatures slightly below 37°C. Lesions in the mouth and pharynx ulcerate quickly because of the absence of a stratum corneum, releasing large amounts of virus into the saliva about the time the cutaneous rash first becomes visible. Virus titers in saliva are highest during the first week of rash, corresponding with the period during which patients are most contagious.

Rudimentary immunologic studies of smallpox were done during the eradication era, but little is known about the immune response to variola major in modern terms. Hemagglutinin-inhibiting (HI) and neutralizing antibodies can be detected beginning about the sixth day of illness or, on average, about 18 days after infection, and complement-fixing (CF) antibodies, approximately 2 days later. ^, Neutralizing antibodies are durable, potentially lasting for life as they have been detected 70+ years after vaccination. The HI antibodies decline to low levels within 5 years, and the CF antibodies rarely persist for longer than 6 months. Little is known about the development of cell-mediated immunity following smallpox, but it likely has a significant part in immunity. Modern studies of the immune response to vaccinia and other orthopoxviruses show a complex array of humoral and cellular immune responses, many with manifestations detectable in the first 3 or 4 days after infection. Immune responses to live attenuated vaccines containing vaccinia virus are likely similar to those developing after variola infection and are discussed in a subsequent section (Immunogenicity of Vaccine).

Except for the lesions in the skin and mucous membranes, other organs are seldom significantly involved in variola transmission. Secondary bacterial infection is not common. Death, when it occurs, probably results from the overwhelming toxemia associated with circulating immune complexes and soluble variola antigens.

Modes of Transmission

Transmission of variola virus, with few exceptions, results from droplets expressed from the oral, nasal, or pharyngeal mucosa of an overtly ill patient that are inhaled by susceptible persons in close contact with the patient. Such transmission is possible from the time of the end of the prodrome and onset of rash and was most frequent during the first week of the exanthem. Spread is generally through large droplets and, thus, was not widely dispersed. ^, Virus also was present in scabs that had separated from the skin lesions of convalescing patients, but epidemiologic evidence shows that infected scabs had a negligible role in transmission of infection, presumably because the virus was tightly bound in its fibrin matrix.

Airborne infection over distances of more than a few meters was uncommon, although two outbreaks within hospitals demonstrated this to be possible as they probably resulted from an aerosol dispersal. ^, In the outbreak that occurred in a hospital in Meschede, Germany, in 1970, a patient initially presumed to have typhoid fever (and cared for under contact isolation precautions) gave rise to 19 other cases of smallpox in patients who had not been in close contact with the index patient. Dissemination in this outbreak was favored by low relative humidity, prevailing air currents within the hospital building, and the presence of cough in the patient. Although cough is not a typical finding of ordinary smallpox, in this case, it would have facilitated viral aerosolization.

There have been instances in which a patient spread the disease to large numbers of contacts, but these were infrequent. In an outbreak in Yugoslavia in 1972, a patient with hemorrhagic smallpox was misdiagnosed as having a severe allergic reaction to penicillin. He was hospitalized because of the development of hemorrhagic disease. In all, he infected 38 healthcare workers and patients before dying. In another patient in the same Yugoslavian outbreak, a woman who had vaccine-modified smallpox spread disease to 16 others. She was a neighborhood matriarch who was visited by many friends and neighbors in her community during her illness and had prolonged face-to-face conversations with many.

Infection of persons such as mortuary workers who handled dead bodies and laundry workers who handled linen from patients has also been repeatedly documented. However, various older accounts that purport to document transmission over great distances on other fomites, such as carpets, letters, and cotton rags, are suspect because the virus does not survive for long periods at customary ambient temperatures or with exposure to ultraviolet sunlight.

Another method of transmission, the ancient practice of variolation (inoculation into the skin of material from pustules or scabs from patients), continued in a number of remote areas until August 1976 and was responsible for many cases in Afghanistan and Ethiopia. In persons inoculated by these methods, extensive rash sometimes developed, and they transmitted infection to susceptible contacts by respiratory droplet infection.

Diagnosis

In endemic areas or when smallpox was known to be circulating, most cases could be readily clinically diagnosed by a history of direct contact with a patient with smallpox, the presence of a prodromal febrile illness, the appearance of a typical deep-seated rash, the centrifugal distribution of lesions, and the fact that all lesions were at the same stage of development on any given area of the body (unlike varicella infection). The infrequent hemorrhagic cases were often initially misdiagnosed as meningococcemia, acute leukemia, or drug toxicity. The diagnosis often was assisted by examination of patients who were the source of their infection or to whom they had transmitted disease. Varicella was by far the most frequent disease confused with the ordinary cases of smallpox ( Table 55.1 ).

Patients with smallpox who had previously been vaccinated and patients with variola minor sometimes had a sparse and atypical rash with minimal systemic symptoms that resembled varicella. In contrast with smallpox, in varicella, the prodromal illness is typically mild or absent, lesions are superficial, appear first on the face or trunk, evolve rapidly and appear to be in different stages of development, and only rarely involve the palms and soles. Severe cases of varicella in adults with extensive rash were also sometimes mistaken for smallpox. For this reason, the Centers for Disease Control and Prevention (CDC) has produced and distributed a chart with an algorithm to assist in the differential diagnosis of acute febrile illness associated with rash. Patients are classified as having a low, moderate, or high likelihood of smallpox based on the presence or absence of a febrile prodrome, the appearance of skin lesions, and other clinical criteria, such as whether the patient appears “toxic.” Only patients reporting a febrile prodrome and who have classic smallpox lesions in the same stage of development are classified in the high likelihood category.

A suspected case of smallpox must be reported immediately to the appropriate local public health authorities, followed by state, province or territorial health departments and, through them, to national authorities and to the WHO. Electron microscopic examination of vesicular or pustular fluid or scabs will reveal the characteristic, brick-shaped orthopox virions. The presence of poxvirus DNA can be rapidly established by PCR testing. Differentiation among variola, vaccinia, and monkeypox is usually apparent from the patient’s history of possible exposure and the growth characteristics of the virus on the chorioallantoic membrane of chick embryos. PCR techniques for rapid identification and differentiation of orthopoxviruses have been developed, ^, and continue to be updated, as new poxvirus strains are sequenced, to provide rapid and reliable disease confirmation for public health responses to outbreaks. These methods are available at the CDC and designated member laboratories of the national Laboratory Response Network. Specimens that are required include vesicular or pustular material (scraped or swabbed from the base of a lesion), punch biopsies, scabs, or venous blood. Detailed specimen collection guidelines are available from the CDC. According to the emergency smallpox response plan available on the CDC website, only personnel immunized against smallpox within the past 3 years or personnel wearing appropriate respiratory and barrier protective equipment should obtain specimens from patients suspected of having smallpox.

Treatment and Prevention With Antivirals

There has been considerable progress in development of antiviral drugs effective against orthopoxviruses in recent years. ^, While a number of compounds have shown some activity against poxviruses (e.g., Apigenin, CGP57380 a MNK1 inhibitor, and small molecule inhibitors of the Golgi-associated retrograde protein complex such as: PA104 and Retro-1), our discussion will focus on those with the most data. Cidofovir and its more bioavailable analog, CMX001, are nucleoside analogs that are active against viral DNA polymerase. They are effective against vaccinia and variola in vitro. Cidofovir is licensed for treatment of cytomegalovirus retinitis, but the administration of cidofovir is complicated by the fact that it is available only for intravenous administration, requires prehydration with saline and probenecid to minimize nephrotoxicity, and requires close monitoring of renal function. CMX001 seems considerably less toxic, but is currently available for human use only as an investigational new drug. Brincidofovir, a lipid conjugate of cidofovir, has been coadministered with both ACAM2000 (a next-generation vaccine based on a clonal derivative of Dryvax) and ACAM3000 (a next-generation vaccine based on the modified virus Ankara [MVA] strain) in a mouse model and found to reduce the severity of vaccination-association lesions with only a slight attenuation in immune response. This drug has also shown promising results in lethal orthopox challenge models in mouse (ECTV, MPXV and VACV) and rabbit (RBPX) models. In a prairie dog model of lethal monkeypox infection, brincidofovir exhibited signs of toxicity at doses higher than 20mg, while low doses of brincidofovir had a partial protective effect, increasing survival from 14% to 57%.

ST-246 (tecovirimat) is a small-molecule compound that targets a gene that produces p37, a major envelope protein required for the production of extracellular virus. ^, Early evidence suggested it is safe for humans, with minimal manifestations of toxicity even at very high doses. It has proved efficacious against several orthopoxviruses in a variety of animal models as a prophylactic agent and in substantially reducing the pathology of established infections. There are animal data that suggest ST-246 can curtail the replication and spread of vaccinia without diminishing the immune response, which would make it ideal for simultaneous treatment and vaccination of close contacts of patients with smallpox (Dennis Hruby, PhD, Oregon State University, Corvallis, personal communication, 2011). ^, Three human patients with immunologic abnormalities and severe life-threatening vaccinia infections have been treated with CMX001 and ST-246. The patients recovered, but were treated with such a wide array of therapies that it is difficult to isolate the specific role of the antivirals used. ^, Ribavirin, another antiviral that has some in vitro activity against vaccinia and variola, was used in a patient with progressive vaccinia along with high doses of vaccinia immunoglobulin (VIG). The role of ribavirin in this patient’s recovery is uncertain. Animal studies using ribavirin to treat cowpox or vaccinia infections have not shown great promise. ^,

Incidence and Prevalence Data

Smallpox is among the most lethal of all diseases. Unlike malaria and other infections that claim millions of lives annually, smallpox occurred globally when it was endemic (i.e., it affected the world’s entire population). In the century before its eradication, it is estimated to have claimed 300 million lives and maimed countless more. As late as the beginning of the smallpox program in 1967, it is estimated that there were 10–15 million cases and 2 million deaths in that year alone. Routine childhood vaccination ceased in 1972 in the United States, ^, and by 1983, most of the rest of the world had followed suit. As a result, vaccine production facilities closed, and although many countries retained their smallpox vaccine stockpiles, those have been gradually depleted. Now, decades after the eradication of smallpox, a large proportion of the world’s population has never been vaccinated or was vaccinated so long ago that immunity has likely waned; thus, the global population is extremely vulnerable to the disease should it reoccur.

The seasonal pattern of smallpox is similar to that of varicella, measles, and other epidemic respiratory diseases. Its incidence was highest during winter and spring. This seasonal peak was consonant with the observation that the duration of survival of an orthopoxvirus in the aerosolized form is inversely proportional to temperature and humidity. Seasonal variation undoubtedly was amplified in many countries by social events, such as the congregation of large numbers of people at festivals and marriage parties during the dry season, and the seasonal movement of nomads.

The age distribution of smallpox cases depended on the immunity of the population, whether the immunity was acquired by vaccination or by infection. Because the disease is less infectious than other childhood diseases such as measles, cases regularly occurred among adults. As recently as 1974–1975 in India, where vaccination was widely practiced and smallpox was endemic, adults constituted 21% of a carefully documented series of 23,546 patients; approximately 2% (or 412) of the patients were older than 50 years. In western Africa between 1967 and 1969, most cases occurred in rural villages, and the age distribution of cases approximated the age profile of the population. Men and women were equally affected in all endemic countries.

The severe Asian form of variola major customarily had overall case-fatality rates that ranged from 40% to 50% for persons younger than 1 year of age and approximately 30% among unvaccinated persons. Variola major in Africa and Indonesia was a somewhat milder disease, with age-standardized case-fatality rates of approximately 10–15%. Variola minor, which after 1967 was present only in Brazil and southern and eastern Africa, resulted in case-fatality rates of 1% or less ( Table 55.2 ).

Most estimates for the R ₀ (the average number of secondary cases infected by each primary case) of smallpox are between 3 and 6, ^, but have ranged as high as 10. A more recent meta-analysis from 2018, found a similar range of 3.5–6 for the R ₀ . Newer reports indicate that vaccine coverage of at least 66–70% is necessary to reach herd immunity levels that will prevent outbreaks, ^, and the Smallpox Eradication Program’s initial goal was to obtain 80% vaccination coverage. ^, However, outbreaks have occurred in populations with high (>90%) vaccine coverage ^, and the first WHO Expert Committee on Smallpox recommended 100% vaccine coverage. Within the household, smallpox was as infectious as varicella but less infectious than measles. With few exceptions, however, smallpox spread less widely and rapidly than these diseases. This finding can be accounted for by the fact that transmission of variola virus does not occur until 2 or 3 days after the onset of the prodromal fever, as attested to by numerous epidemiologic observations. By then, most patients already were confined to bed because of the high fever and malaise, and, thus, secondary cases usually were restricted to the few who came in contact with patients in the household or hospital. For this reason, smallpox outbreaks tended to be clustered in a segment of a town or village and in localized areas of a province or district.

High-Risk Groups

With routine immunization being stopped in nearly every country by 1983, nearly all children and the majority of adults have little or no protection against smallpox. If smallpox were to be released today, vaccination would be indicated for all people considered to be at immediate risk of exposure. The people at highest risk would be healthcare providers who might have contact with patients before the disease has been diagnosed and laboratory workers, and those who had never received a dose of smallpox vaccine.

Reservoirs of Infection

Variola virus is a uniquely human pathogen and since its eradication in 1980, there are no reservoirs of infection. Today, there is concern that smallpox virus could be released accidentally from a laboratory or deliberately as a biological weapon. Although the risk of these events is not quantifiable, they are generally acknowledged as possible. There are also concerns regarding outbreaks of other zoonotic orthopoxviruses such as monkeypox, and, indeed, a monkeypox outbreak occurred in the midwestern United States in 2003 as a result of the exotic small pet mammal trade. Fortunately, it was quickly recognized and dealt with appropriately.

Smallpox as a Biological Weapon

There are anecdotal reports that Spanish explorers gave Amerindians contaminated clothing, leading to devastating epidemics among the Aztecs and Incas, among others. During the French and Indian Wars (1754–67) the British General Lord Jeffery Amherst suggested to his staff that they distribute smallpox-infected blankets to Amerindians. It is not certain that they were successful, however, as smallpox was already raging among some tribes in the Hudson and St. Lawrence River valleys at the time. During the Revolutionary War, smallpox became a serious problem for the Continental Army, forcing George Washington to institute mandatory variolation for all American soldiers.

The media have raised concern about the risk of a terrorist biological attack using smallpox. Such risks are unknown but probably quite low. Much of the concern stems from a book written by the former deputy director of the Soviet Union’s BioPreparat that states that during the height of the Cold War there existed manufacturing facilities capable of producing tons of smallpox virus and that the State Research Center of Virology and Biotechnology (the VECTOR Institute), Novosibirsk, Russia, pursued the possible use of recombinant products of smallpox virus as biological weapons. There have been no public statements by nation states or terrorist groups about development of smallpox as a bioweapon. The United States did not find evidence that smallpox virus existed in Iraq, despite the claims of the aforementioned book’s author, that Saddam Hussein had the means and plans to use smallpox as a weapon of mass destruction. No published evidence suggests that smallpox is being illegally produced anywhere else. Furthermore, efforts to recover virus from bodies buried in the tundra or from mummified corpses have been unsuccessful. ^,

In terms of weaponization, variola virus may be lyophilized and is relatively stable as an aerosol. The infectious dose, as suggested from the epidemiology of a hospital outbreak in which aerosol spread was documented, may be only a few virions and may be effectively disseminated through aerosolization. The resulting disease has high mortality and morbidity rates. The current world population is immunologically naïve or has waning levels of protection. Vaccinia immune globulin and a number of antiviral drugs are available to treat the disease, but these treatments have never seen wide scale use to control smallpox outbreaks. It is likely that even a small outbreak would cause global panic and disruption.

Since the terrorist events of September 11, 2001, and the anthrax attacks of Fall 2001, the U.S. federal government has procured a large stock of vaccine to meet the needs of the nation in case of an outbreak of smallpox. As of 2015, the U.S. Strategic National Stockpile contained three smallpox vaccines: (1) sufficient doses of ACAM2000 to vaccinate the entire U.S. population, (2) 85 million remaining doses of Aventis Pasteur Smallpox Vaccine (APSV, or “wetvax,” a liquid calf-lymph product), and (3) more than 20 million doses of Imvamune (a modified vaccinia virus Ankara [MVA]-based vaccine) to be used in populations for whom live vaccinia vaccines are contraindicated. Studies indicate that both APSV and Imvamune can be diluted up to fivefold and retain immunogenicity. In 2002, the CDC released a guidance plan to public health agencies detailing the necessary steps to be taken to contain an outbreak of smallpox, including vigorous surveillance for cases, rigorous isolation of cases, and identification and vaccination of close contacts. Nevertheless, most experts believe that a deliberate release of smallpox remains a low-likelihood but high-consequence event.

History of Vaccine Development

Although early smallpox vaccines were cowpox virus, a virus from a disease called horsepox (HSPV) was sometimes used. Eventually, however, vaccinia virus became the poxvirus of choice in most major vaccines. Although its exact origins are unclear, vaccinia is not a mutant of variola virus; rather, it may have arisen as a hybrid of cowpox and another orthopoxvirus or through thousands of serial passages under artificial conditions of culture. It is also possible that the species represents a laboratory survivor of a now naturally extinct species of orthopoxvirus. Characterization of strains is further complicated by the fact that a seed lot system for vaccine production was not used widely until the 1960s. Thus, even the strains with common names and ancestors have different passage histories, having been passed sequentially through a variety of vaccinifers, such as cows, sheep, and water buffalo, with periodic passages through rabbits, horses, and even humans. Indicative of the scientific ignorance of vaccine technology until recent decades is a statement of the Ministry of Health of Great Britain, which, in 1928, advised that seed lymph could be obtained from (a) “smallpox direct”; (b) cowpox; (c) horsepox, sheep pox, or goatpox; and (d) vaccinia in the human body. At least one smallpox vaccine produced in the United States in the early 1900s is genetically similar to horsepox, providing additional evidence that multiple poxviruses were used in smallpox vaccines. Five orthopox virus genomes were identified using materials from vaccination kits from the American Civil War era. Interestingly, these viral genomes aligned closely with the previously mentioned HSPV.

At various times, attempts were made to create vaccine products based on inactivated virus. Inactivated vaccines elicited high titers of neutralizing antibodies but were ineffective at preventing disease in animal models and human populations. Inactivated vaccines consist almost entirely of a single type of poxvirus virion: mature virus (MV) particles, which lack an additional membrane, and associated proteins found on the other major type of poxvirus virion, extracellular virus (EV) particles. Consequently, these vaccines are unable to induce responses to EV proteins, a defect that likely accounts for their lack of protective efficacy.

Attenuated strains of vaccinia were created in an effort to diminish the incidence of serious adverse events. Several of these strains based on the New York City Board of Health (NYCBOH) strain (Rivers, CVI-78) produced less-severe reactions but also had reduced immunogenicity, calling into question the level of protection provided against smallpox. Another attenuated vaccine, the MVA strain, was produced by a German research group through more than 500 passages in chick embryo fibroblast cells. ^, During passage, multiple genetic deletions occurred (approximately 15% of its genome), and it became much more narrowly restricted in its host range. Laboratory studies demonstrated unimpaired MVA gene expression in human cells and a block in virion morphogenesis. The ability to achieve high expression of recombinant genes despite abortive replication is a significant characteristic of this mutant virus. MVA was only developed during the latter part of the eradication effort and, consequently, was never used in a smallpox-infected area. Nevertheless, the potentially improved safety profile of MVA and other attenuated vaccinia strains has led to renewed development of attenuated smallpox vaccine candidates and their clinical testing and licensure (see “Future Vaccines” later).

Development of the Smallpox Vaccine

A method for protection against naturally acquired smallpox infection seems to have been discovered in India sometime before 1000 AD, although there is evidence for its use in China as well. There it became the practice to deliberately inoculate, into the skin or by nasal insufflation, scabs or pustular material from lesions of patients. This practice (now called variolation) resulted in an illness usually less severe than that acquired naturally by inhalation of droplets. From India, variolation spread to China, western Asia, Africa, and finally, in the early 18th century, to Europe and North America. ^, Case-fatality rates associated with variolation were considerably lower than fatality rates with naturally acquired infection, but infected persons were still capable of transmitting smallpox to others. After cowpox (an orthopoxvirus closely related to variola virus) began to be used as a protective vaccine, the practice of variolation diminished, but continued to be practiced in Ethiopia, western Africa, Afghanistan, China, and Pakistan as recently as the 1960s and 1970s.

Variolation was gradually replaced with vaccination. In fact, within 5 years of publication, Jenner’s seminal paper had been translated into six other languages and vaccination was widely used in many countries of Europe. Within a decade, this process began to be used in countries throughout the world. Historically, the chronicles of the de Balmis expedition of 1803–1806 vividly describe the transport of the vaccine by sea to Spanish colonies in the Americas and Asia by serial arm-to-arm vaccination of orphaned children. ^,

As the 19th century progressed, the initial wave of enthusiasm for vaccination subsided. Occasionally, syphilis, hepatitis, and other infections were transmitted in the process of arm-to-arm vaccination. ^, In some areas, significant opposition occurred among religious and governmental leaders and antivaccinationist societies that opposed the principle of infecting humans with an animal disease, especially under civil mandate. Despite Jenner’s claims of lifelong immunity, smallpox can and did occur in patients who had previously been successfully vaccinated, and the need for revaccination was demonstrated early in the 19th century. Other issues included difficulty obtaining fresh vaccination material and difficulty transporting vaccine over long distances. These problems were eventually solved by growing vaccinia on the flanks of calves or other animals, providing an adequate and safer supply of vaccine material. This approach was initially used in Italy as early as 1805 but did not become widespread until the mid-1800s. ^, In the 18th century an English physician, John Haygarth, carefully studied local outbreaks and disease transmission, then published simple rules for the prevention of smallpox. These rules (avoid entering homes of infected individuals, quarantine yourself when sick, clean everything that comes into contact with the patient, do not allow patient to approach susceptible people) proved to be highly effective when followed.

Vaccine Use and Disease Occurrence in the United States

In the United States, variolation was promoted by a prominent minister, Cotton Mather, in collaboration with a Boston physician, Zabdiel Boylston. ^, General George Washington instituted variolation among the Continental Army in 1777 in an effort to minimize the drastic effect smallpox outbreaks were having on soldiers’ combat readiness. Thomas Jefferson and his family underwent variolation in 1782. Benjamin Franklin lost a 4-year-old son to smallpox in 1736 and later became an outspoken proponent of variolation, even setting up an organization to provide free variolation to poor people. ^,

Benjamin Waterhouse performed the first vaccinations in Boston, Massachusetts, in July 1800 using material provided by Jenner and is widely credited with introducing vaccination to the United States. ^{, , ,} President Thomas Jefferson also actively promoted vaccination, although its routine use did not occur until the 1900s owing to difficulties sustaining the virus through arm-to-arm inoculation. Epidemics of variola major continued to occur at intervals, depending on population density and frequency of importations.

Although outbreaks of variola major continued to occur until about 1927, most 20th-century cases of smallpox in the United States were caused by variola minor. The origin of this strain is unknown. However, a similarly mild strain of smallpox, called Kaffir pox, had begun to circulate widely in South Africa in the early 1890s. What, if any, connection there was between these two foci of variola minor infections is unknown. Because the disease was mild and the case-fatality rate was less than 1%, interest in vaccination waned and smallpox incidence increased sharply. To control the disease, public health authorities sought to compel vaccination as a requirement for school entry, an action upheld by the U.S. Supreme Court. However, antivaccinationist sentiment and antipathy toward compulsory measures prevailed in many states, most of which did not support the legislation or actually prohibited compulsory vaccination. Reported cases of smallpox declined from 108,487 in 1921 to 30,151 in 1931. Between 1932 and 1939, 5,000–15,000 cases were reported annually, with 21–52 deaths. During the following decade, reported U.S. cases steadily diminished, with the last occurring in 1949. This progress occurred in the absence of a nationally coordinated smallpox control effort. Leake attributed improved smallpox control and its eventual elimination to the wider availability of refrigeration and, consequently, better preservation of the vaccine. Routine childhood vaccination continued in the United States until 1972 as a protection in case smallpox was imported and was enforced in most states by compelling vaccination for school entry. In the early 1960s, many importations of smallpox occurred in Europe; almost half of all subsequent cases occurred as a result of contact in hospitals. Accordingly, beginning in the 1960s, the CDC urged the routine vaccination of hospital staff, but few hospitals complied. After the global eradication of smallpox, distribution of vaccine was restricted to the military and to the few laboratories working with orthopoxviruses.

Vaccine Use and Disease Occurrence in the Industrialized World

Of the other major industrialized countries, Canada interrupted transmission of smallpox in the early 1940s, and Japan about 1950. Vaccination continued in all industrialized countries, as it did in the United States, until the mid to late 1970s, as a protective preventive measure in case smallpox was reintroduced. Australia and New Zealand were notable exceptions. These countries, protected by distance and strict quarantine measures, never vaccinated widely but also never became endemic for smallpox.

In 1958, a WHO study group first recommended that a seed lot system be used in vaccine manufacture. Beginning in 1967, an increasing number of vaccine producers began to use one of two strains. Most common was the Lister strain from the Lister Institute in England, which was propagated as seed virus by the National Public Health Institute of The Netherlands for distribution by the WHO. The second strain was the NYCBOH strain, propagated by Wyeth Laboratories, Radnor, PA, using the trade name Dryvax. Three of the largest countries, Russia, China, and India, used other strains: EM-63 in the Soviet Union, the Temple of Heaven strain in China, and the Patwadanger strain in India.

Vaccine Use and Disease Occurrence in the Developing World

With an assured source of vaccinia, the numbers of vaccinations in Europe increased and smallpox was largely controlled in Europe and North America by the end of World War II. Unfortunately, the disease remained endemic throughout tropical and semitropical countries, in part because the vaccine was heat-labile and difficult to keep viable in tropical areas. It had long been noted that dried vaccines were more stable than liquid formulations. Two of the more common methods involved (a) placing vaccine lymph with sulfuric acid under vacuum conditions and allowing it to dry at room temperature and (b) mixing vaccine lymph with stabilizing and antibacterial agents such as peptone and phenol, followed by centrifugal freeze-drying techniques originally pioneered for human plasma. In the 1940s, techniques were developed for large-scale and commercially feasible processes for the manufacture of stable, freeze-dried smallpox vaccine, leading to increased success with vaccination and eradication programs in Indonesia and parts of Africa.