Review

Poxviruses

VARV belongs to the genus Orthopoxviridae, family Poxviridae, that includes the agents of VACV, MPXV, cowpox, camelpox and ECTV. Members of Poxviridae have large linear double-stranded DNA, with genome sizes ranging from 130 to 300 kbp. Most of the essential genes for poxvirus replication and survival are located in the middle of the genome, and tend to be conserved across the family.⁹ Poxviridae is classified into two subfamilies, Chordopoxvirinae and Entomopoxvirinae. Chordopoxvirinae include the genera Orthopoxviridae, Avipoxviridae and several others, as outlined in Table 1. Molecular analysis of DNA polymerases has suggested a possible eukaryotic origin of poxviruses.¹⁰ Indeed, the use of sophisticated machinery to replicate in the host cytoplasm also supports this hypothesis of poxvirus origins. The biology and genetics of poxviruses is explored in greater detail in two excellent chapters.^{9, 11}

Table 1 - Host restriction of selected poxviruses of vertebrates.

Full table

There is little cross-reactivity between the different genera of Chordopoxvirinae; however, there is evidence of cross-reactivity and cross-protection within a genus. Both in in vitro tests and in experimental animals, members of the orthopoxvirus genus exhibit extensive serological cross-reactivity. Indeed, the considerable cross-protection afforded between orthopoxviruses was exploited in the use of VACV to vaccinate against VARV for the eradication of smallpox.

In addition to VARV, other poxviruses are also able to infect humans, although none has caused as much devastation as VARV. Other disease-causing poxviruses include molluscum contagiosum virus, VACV and MPXV.^{1, 12}

Smallpox: the disease

There are two principal forms of the smallpox, variola major and a much milder form, variola minor (alastrim). Variola major was the dominant form of smallpox worldwide till the end of the 19th century. At the turn of the century, variola minor was first detected in South Africa and later in Florida, from where it spread across the United States and into Latin America and Europe. Variola major epidemics such as those that occurred in Asia resulted in case-fatality rates of 30% or higher among those unvaccinated, whereas in variola minor rates were about 1% or less.¹ In the past, clinical differentiation of these two forms was possible only during outbreaks, but today, with modern diagnostic methodologies, virological differentiation is possible.¹³

Smallpox spreads from person to person primarily by droplet nuclei or aerosols expelled from the oropharynx of infected persons, and by direct contact. VARV in an aerosol suspension can spread widely and infect at very low doses.¹⁴ Contaminated clothing or bed linens can also spread the virus.

After an incubation period that usually lasts between 10 and 14 days, the illness is heralded by a febrile prodrome 1–4 days before a rash erupts. Thereafter, an abrupt onset of high fever follows, accompanied by constitutional symptoms like headache, backache, chills, abdominal pain and vomiting. Lesions erupt in the oropharynx, mouth and tongue, and about 24 h later a rash develops. Commonly the skin lesions develop first in the face and spread to all parts of the body within a day or two. The rash is characteristically macular at the outset, but progresses to papules, then vesicles and forms pustules within a week. In 2–3 weeks these pustules become crusted. The many complications of smallpox include bacterial infections of skin, joints, bones, lungs and other organs and the dreaded generalized sepsis. Smallpox can also lead to corneal ulceration, arthrits, keratitis, pneumonia and encephalitis.^{1, 15}

The immune response in smallpox

Our understanding of the host response to poxvirus infection in humans comes from historical clinical data on smallpox patients and vaccinated individuals. Although the smallpox vaccine was highly effective in the vast majority of the population, it was a live virus vaccine. In some immunodeficient patients therefore, exposure to it resulted in severe generalized VACV infection. Intriguingly, individuals with abnormalities of T-cell function developed generalized vaccinia, whereas patients with congenital agammaglobulinemia did not.¹ Furthermore, immunoglobulin therapy for generalized vaccinia was thought to be effective only through its ability to control virus long enough to allow the restoration of cell-mediated immunity. This led to the view that antibody did not significantly contribute to recovery from a primary poxvirus infection and that it was T-cell function that was critical in virus control. However, as discussed below, there is now mounting evidence that antibody plays a crucial role in recovery from poxvirus infection in several animal models.

A correlative link between the presence of serum antibody and protection from smallpox can be drawn from historical clinical data.^{16, 17, 18} Downie and McCarthy¹⁶ reported in 1958 that neutralizing antibody was either absent or present only at very low titers in sera from fatal cases of smallpox. Another prospective study on patients in Lahore found that all contacts of smallpox patients who had a neutralizing antibody titer of above 1:32, were protected against smallpox, although some contacts with no detectable antibody were also spared.¹⁷ In contrast, a study on contacts of smallpox patients in Calcutta by Sarkar et al.¹⁸ found that although none of the vaccinated contacts developed disease, this was regardless of their antibody levels.

As early as 1913, a monograph entitled 'Studies in small-pox and vaccination' by William Hanna reported that although immunity to smallpox waned over time, 93% of the people vaccinated against smallpox were protected from disease for over 50 years. More recent studies have confirmed that specific immune parameters are long-lived after smallpox vaccination. El-Ad and co-workers found that despite an initial decline in the first 3 years after vaccination, antibody levels remain stable for at least 30 years after the last re-vaccination.¹⁹ It has also been reported that T-cell immunity remains stable for decades.²⁰ B-cell memory to smallpox has been found to persist for 50 years in one study²¹ and 90 years in another,²² after vaccination with the DryVax vaccine. Another study showed that serum antiviral antibody responses remained stable between 1–75 years after infection whereas antiviral T-cell responses weakened over time, with a half-life of 8–15 years.²³ Further dissection of the post-vaccination, antigen-specific T-cell responses showed that a robust Th-1-biased, CD4T cell response can be detected following vaccination and is stable during the contraction phase (between peak effector and memory phases of the immune response). In contrast, CD8T cell memory declines more rapidly.²⁴ Collectively, these studies strongly suggest that antibodies play a crucial role in the long-term protection from smallpox and that T cells may also play an important role.

Ectromelia virus

Our current understanding of immunity to poxvirus infection benefited enormously from studies on the response to VACV vaccination in humans and from animal studies using VACV and closely related orthopoxviruses, such as MPXV and ECTV. The need for surrogate small-animal models of smallpox is amplified by the fact that VARV has a restricted host range and is known to infect only humans. Experiments with VACV in mice and MPXV in macaques utilize very high doses of virus to induce disease that mimics the situation in smallpox. Consequently, the disease produced is limited to representing the post-secondary viremic stage of smallpox in humans, bypassing the natural progression of pathogenesis. ECTV, on the other hand, encodes many immune-modifying proteins similar to VARV, is highly virulent and infectious at very low doses, causing disease, termed mousepox, with high mortality rates, and shares many aspects virus biology, pathology and clinical features of smallpox. Laboratory mouse models are the most versatile, in which the roles of individual components of innate and adaptive immunity can be investigated since there are many well-characterized inbred strains as well as gene-deficient mice that allow for careful dissection of the key immune parameters. For these reasons, the mousepox model is arguably the best surrogate for VARV.^{1, 25} Nevertheless, one difference between the two diseases is that in smallpox, the cause of death was frequently unknown and sometimes associated with toxemia (septicemia), with little virus involvement in the liver. On the other hand, the cause of death in mousepox is associated with a high viral load in most visceral organs and in particular the liver.

Similar to the existence of a number of strains of VARV, there are different strains of ECTV, which have been used for experimental purposes, each possessing a different degree of virulence in their natural host, the mouse. The Moscow, Hampstead and NIH 79 strains have been the most studied, the Moscow strain being recognized to be the most virulent and infectious strain for mice.^{26, 27}

ECTV causes a generalized disease termed mousepox in mice.²⁸ The virus has co-evolved with its host and as part of its survival strategy, encodes many host response modifiers (reviewed in Seet et al.²⁹ and Smith and Alcami³⁰), which counteract the effects of various components of the antiviral host immune response. In addition, the fact that infection with ECTV in mice rapidly becomes systemic when inoculated via the subcutaneous route has been exploited by immunologists who have used this model extensively, not only as a tool to investigate the pathogenesis and immunology of poxvirus infections, but also as a model of generalized virus infections.³¹

Genetic resistance in mousepox

Early studies have identified that resistance to mousepox depends on the route of infection, virulence of the infecting virus and the genotype of the mice.^{32, 33, 34} Inbred strains of mice can be categorized as resistant or susceptible to ECTV infection. The susceptible mouse strains BALB/c, A and DBA/2 are unable to control virus replication and spread, and usually die from uncontrolled liver necrosis. The resistant strains such as AKR, C57BL/6 (B6) and some strains of 129 mice, on the other hand, have very limited pathology and low mortality rates.^{35, 36} In this context, it is important to note that the outbred human population also had a range of susceptibilities to virulent strains of VARV, and although mortality rates associated with smallpox were high, a significant subset of the infected population recovered. The basis of susceptibility or resistance, and the genetic factors associated with recovery from VARV infection in humans, has not been elucidated.

In the mousepox model, there is a general consensus that resistance in inbred mouse strains is controlled by multiple unlinked autosomal dominant genes that map to at least four gene complexes. The resistance to mousepox (Rmp-1) locus on chromosome 6 is known to control replication of virus in the liver and maps to the natural killer gene complex (NKC), which functions in signal transduction and activation of natural killer cells.^{37, 38} The second locus, Rmp-2, is located on chromosome 2 and maps to a region near the complement component C5 gene. In response to ECTV infection, this locus protects female mice more than males.³⁷ At the site of infection, C5-deficient mouse strains, such as DBA/2, have impaired recruitment of circulating leukocytes. Rmp-3, on the other hand, is linked to the major histocompatibility complex (MHC) (H-2)³⁹ and expresses its function via CD8T cells, which have been shown to be critical for recovery from mousepox.^{40, 41} Rmp-4, believed to encode a gonadal-dependent function,^{37, 42} has been mapped to the selectin gene complex.^{37, 42} Mechanisms determining innate resistance to ECTV appear to act early after infection as susceptible strains begin to demonstrate signs of illness as early as day 3 post-infection (p.i.) and, depending on the dose and route used, succumb to mousepox as early as day 6 or 7 p.i.⁴¹

Pathogenesis of ECTV infection

When introduced via the subcutaneous route, ECTV multiplies locally, and then spreads to and multiplies in the draining lymph node. Invasion of the bloodstream initiates a primary viremia that allows the virus to invade the spleen and liver, where massive cell necrosis next liberates virus into the bloodstream, causing a secondary viremia. This secondary viremia is responsible for seeding the skin, causing the characteristic pock lesions of mousepox. These lesions, which parallel those found during smallpox in humans, continue to seed the skin with infectious viral particles, and are an important route of transmission of the disease. Death in the susceptible strains of mice is usually due to massive hepatic necrosis⁴³ and usually occurs around day 8–10 p.i. This coincides with the time at which viral titers are highest in these mice.

Immune response to ECTV

The immune response can be broadly divided into the innate and the adaptive phases, distinct but complementary and interactive parts. Upon viral entry, the largely nonspecific innate phase is initiated rapidly to protect the host early during the infection, and prevents the infection from being established, while awaiting the slower onset of the antigen-specific adaptive response. Immunity to ECTV and recovery from mousepox are heavily dependent on various facets of both the innate and the adaptive immune responses. These include the effector functions of natural killer cells, CD4 and CD8T lymphocytes, macrophage subsets, nitric oxide, T helper type 1 cytokines and interferon- (IFN-), - and -.^{40, 44, 45, 46, 47, 48, 49} More recently, it has been recognized that B cells and antibody are critical in the control and recovery from ECTV infection.^{50, 51}

Role of antibody in a primary ECTV infection.

Much of the emphasis in trying to elucidate the immune response to poxvirus infections was for a long time focused on the critical contribution of T-cell effector function,³¹ to the neglect of B-cell function. Early indications that there might be a role for B cells and antibody came from T-cell depletion studies in ECTV-resistant B6 mice.⁴⁰ Monoclonal antibody-mediated CD8T cell subset depletion of ECTV-infected B6 mice abrogated the CTL response, rendering these mice unable to clear virus. The mice were thus overwhelmed by the massive viral load, and exhibited 100% mortality by day 10 p.i. On the other hand, CD4T cell depletion resulted in a suboptimal CTL response, poor control of virus and virus resistance.⁴⁰ An important role of CD4T cells is to provide help for CTL function; therefore, a suboptimal antiviral CTL response in mice lacking CD4T cells was expected.⁴⁰ However, in contrast to control mice, CTL activity in these mice persisted even in the late stages of infection, but this response was not sufficient to clear virus. Similar findings were observed in mice deficient in MHC class II (that also lack CD4T cells), which also displayed virus persistence in major organs even at day 26 p.i. Unlike animals lacking CD8T cells that died early, these mice developed pock lesions on the skin by about day 12–14 p.i. Neither the mice deficient in CD4T cells nor MHC class II were able to generate neutralizing antibody,⁵⁰ as these animals lacked the capacity for CD4T cell interaction with B cells.

To directly investigate the requirement for B cells and antibody in ECTV infection, we utilized B-cell-deficient (MT^-/- mice).⁵² These B-cell-deficient mice were susceptible to primary ECTV infection, and demonstrated 100% mortality within 3 weeks p.i., despite mounting a normal CTL response to the virus. Through the course of infection, MT^-/- mice developed skin lesions on the tails and ears. The lesions, which developed as papules, progressed with time to ulcers. Passive transfer of ECTV-immune serum or transfer of B cells from naïve wild-type mice, but not B cells from MHC class II deficient mice, facilitated virus clearance and complete recovery of MT^-/- mice from ECTV infection.⁵⁰ Intriguingly, all the skin lesions completely healed in mice that recovered. This finding parallels the observations in smallpox, where pock lesions completely healed in patients who recovered. This series of experiments demonstrated the role for B cells and antibody in ECTV clearance. Our results are consistent with those of Fang and Sigal,⁵¹ who also found that CD8T cell responses alone are insufficient to control mousepox at the later stages of infection. The fact that mice deficient in CD8T cell function die early in infection, whereas those deficient in B cells or antibody production die much later, indicates that B-cell function becomes critical after the effector phase of the CD8T cell response to infection subsides.

Role of antibody in a secondary ECTV infection.

As outlined above, prospective studies on vaccinated contacts of smallpox patients support a role for antibody in protection from smallpox. Using the mousepox model, we have shown that poxvirus control and recovery from secondary infection is absolutely dependent on the generation of neutralizing antibody.^{47, 53}

For these studies we employed a prime-challenge approach, where mice were first primed with a thymidine kinase-deficient, avirulent strain of ECTV⁵⁴ that replicates less efficiently than the wild-type virus, but induces both cell-mediated and antibody responses, both of which are undetectable by 4 weeks p.i.. Five to six weeks later, mice were challenged with wild-type ECTV. It should be noted that normally susceptible strains of inbred mice, such as BALB/c, that succumb to infection with wild-type ECTV can mount a successful immune response to wild-type ECTV and clear virus if they are first primed with the thymidine kinase-deficient, avirulent strain in this regime.

MHC II^-/-, CD40^-/- and MT^-/- mice, normally susceptible to wild-type ECTV infection, recover completely from the thymidine kinase-deficient, avirulent strain of ECTV. However, when infected with wild-type ECTV in a secondary challenge, these mice fail to mount a neutralizing antibody response, have poor virus control and succumb to infection in a manner similar to those animals that have not been primed with avirulent virus. This strongly indicates that the presence of B cells, coupled with CD4T cell–B-cell interaction (absent in CD40^-/- and MHC II^-/- mice) and subsequent antibody production, is important for recovery from a primary⁵⁰ as well as a secondary infection.^{47, 53}

Unlike the response to a primary poxvirus infection, where a number of immune parameters including CD4 and CD8T lymphocytes, IFN-, - and - and neutralizing antibody^{40, 44, 45, 46, 47, 48} all play a critical role; an effective response to a secondary infection is dependant primarily on a B-cell response and virus-specific antibody.^{47, 53}

The finding that IFN function was redundant for recovery from a secondary ECTV infection was unexpected,⁴⁷ as its requirement in combating a primary virus infection is undisputed. The significance of IFN in the host response is further underscored by the fact that ECTV, a natural mouse pathogen that has co-evolved with the host, itself encodes viral homologs of secreted receptors for IFN-/³⁰ and IFN-.^{55, 56} This is not unique to ECTV as VARV and several other poxviruses also encode receptor homologs that bind to host IFN and modulate their signaling pathways.⁵⁵ Using the prime-challenge regime described above, we showed that mice deficient in type I and/or type II IFN function, or IFN regulatory factor 1, are able to effectively control virus and recover from a secondary ECTV infection.⁴⁷ These animals generate a neutralizing antibody response even in the absence of IFN function; although when present, IFN strongly influences the neutralizing titer and subtype of IgG that is produced.

The contribution of CD8T cell function is mandatory for control of a primary poxvirus infection, and mice deficient in IFN-, perforin, granzymes A and B, perforin and granzymes A and B, or 2-microglobulin that lack CD8T cells, are all highly susceptible to primary ECTV infection.^{40, 45, 57, 58, 59} However, these animals, as well as wild-type B6 mice depleted of CD4, CD8 or both subsets, are fully protected from a secondary infection. Protection correlates with effective virus control and generation of neutralizing antibody.

The finding of a neutralizing antibody response in the absence of CD4T cell help,^{47, 53} although not in keeping with current dogma, is supported by several recent studies.^{60, 61} It would be interesting indeed to dissect the mechanism of this phenomenon, as it has the potential to have widely ranging implications in therapeutics and vaccine design, especially in circumstances where immunodeficiency precludes the use of certain available vaccines.

Antibody responses to other poxviruses

There is also a growing appreciation of the importance of antibody in virus control and recovery in other models of both primary and secondary poxvirus infections. There is evidence that myxoma virus infection in rabbits elicits a protective antiviral antibody response.⁶² Xu et al.⁶³ have utilized a mouse model of VACV infection to show a role for antibody in the control of virus replication in a primary infection, but only if CD4 or CD8T cells were present. Requirement for antibody in protection against re-infection is more established, and support for this comes from several different models. A study on MPXV infection of macaques demonstrated that VACV vaccine induced protection against a lethal intravenous challenge.⁶⁴ This was lost if the animals were depleted of B cells, but not if they were depleted of either CD4 or CD8T cells. This is in agreement with the data from the mousepox model.⁵³

Many studies have used VACV as the prototypic orthopoxvirus and as such, there is significantly more information about this virus. The infectious virus particles exist in two major forms. The extracellular enveloped virus is composed of the intracellular mature virus wrapped in a double membrane acquired from the Golgi apparatus.⁶⁵ Both forms of the virus can be neutralized by antibody.^{66, 67} However, antibody formed against the extracellular enveloped virus cannot neutralize intracellular mature virus and vice versa. Among VACV proteins, at least five viral products, encoded by genes H3L,⁶⁸ A27L,⁶⁹ B5R,⁷⁰ D8L⁷¹ and L1R,⁷² are known to contain epitopes that elicit neutralizing antibodies. Antibodies are formed against the B5 protein of the extracellular enveloped virus, and immunization of mice with the B5 protein protected against a lethal intranasal VACV challenge.⁷⁰ The A27 gene of VACV encodes a 14-kDa protein, which forms covalently linked trimers localized in the intracellular mature virus form.⁷³ When this protein interacts with the 21-kDa protein (product of the A17 gene), a trimer is formed. The C3 monoclonal antibody raised against this protein has virus-neutralizing activity in vitro,⁶⁹ and immunization of mice with the purified form of this protein confers protection from a lethal VACV challenge.⁷⁴ A smallpox DNA vaccine encoding four VACV genes (L1R, A27L, A33R and B5R) protects against a lethal challenge of MPXV.⁷⁵ A more recent study showed that immunization with the DNA vaccine plus the four peptides improves upon protection afforded by the vaccine alone.⁷⁶

A study by Belyakov et al.⁷⁷ has showed that in the absence of B cells, vaccinated mice challenged with virulent VACV contract disease, alluding to a role for antibody, although no mortality was noted. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. In another study, mice with immune deficiencies were protected against a lethal VACV challenge.⁷⁸ These studies with VACV serve to emphasize the point that although VACV and ECTV are genetically very similar, the requirements for recovery from infection are different. Perhaps this disparity hinges on the fact that the natural host for VACV is unknown, whereas the natural host for ECTV is the mouse. Hence, one has to be cautious when considering findings made with VACV infection of mice and their implications for poxvirus immunity.

Finale: significance of antibodies in poxvirus infections

Although early studies in smallpox patients and vaccines alluded to a role for antibodies, the critical role of antibody in resolving poxvirus infection has received more recognition in the present day. The growing evidence for a requirement for antibody in protection from both a primary as well as a secondary poxvirus infection, has disrupted the stronghold of the T-cell school, which for so long dominated poxvirus immunology research.

It is apparent now that antibodies, along with T cells and other arms of the innate immune system, need to be considered as targets of new generation vaccines for poxviruses. The journey is but half over, as many different avenues continue to be explored, with the ultimate goal of identifying, as accurately as possible, the correlates of protection against smallpox. The continuing revolution in molecular biology, genomics, proteomics and in cross-disciplinary fields, such as systems biology and mathematical modeling has allowed the exploration of more sophisticated tools to help us understand the poxviruses, and promises to deliver new findings, which will hopefully crystallize in more efficacious therapeutic interventions and vaccines.

Notes

Declaration

The authors declare no conflicting financial interests.

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