Revisiting Jenner's mysteries, the role of the Beaugency lymph in the evolutionary path of ancient smallpox vaccines.

In 1796, Edward Jenner developed the smallpox vaccine consisting of pustular material obtained from lesions on cows affected by so-called cow-pox. The disease, caused by cowpox virus, confers crossprotection against smallpox. However, historical evidence suggests that Jenner might have used vaccinia virus or even horsepox virus instead of cowpox virus. Mysteries surrounding the origin and nature of the smallpox vaccine persisted during the 19th century, a period of intense exchange of vaccine strains, including the Beaugency lymph. This lymph was obtained from spontaneous cases of cow-pox in France in 1866 and then distributed worldwide. A detailed Historical Review of the distribution of the Beaugency lymph supports recent genetic analyses of extant vaccine strains, suggesting the lymph was probably a vaccinia strain or a horsepox-like virus. This Review is a historical investigation that revisits the mysteries of the smallpox vaccine and reveals an intricate evolutionary relationship of extant vaccinia strains.

Defending against smallpox: a focus on vaccines.

Smallpox has shaped human history, from the earliest human civilizations well into the 20th century. With high mortality rates, rapid transmission, and serious long-term effects on survivors, smallpox was a much-feared disease. The eradication of smallpox represents an unprecedented medical victory for the lasting benefit of human health and prosperity. Concerns remain, however, about the development and use of the smallpox virus as a biological weapon, which necessitates the need for continued vaccine development. Smallpox vaccine development is thus a much-reviewed topic of high interest. This review focuses on the current state of smallpox vaccines and their context in biodefense efforts.

The evolution of poxvirus vaccines.

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

Position on donors and smallpox vaccine: a committee opinion.

Although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, SART/ASRM accordingly recommends that ART practitioners consider deferring donors who have recently received smallpox vaccine or contracted symptomatic vaccinia virus infection through close contact with a vaccine recipient (until after the vaccine or infectious scab has spontaneously separated). Good donor practice further suggests that donors who are not in good health, including those with recent complications from smallpox vaccine, should be similarly deferred. (This document was reviewed by the ASRM Practice Committee in 2011).

The identification of HLA class II-restricted T cell epitopes to vaccinia virus membrane proteins.

Three decades after the eradication of smallpox, the threat of bioterrorism and outbreaks of emerging diseases such as monkeypox have renewed interest in the development of safe and effective next-generation poxvirus vaccines and biodefense research. Current smallpox vaccines contain live virus and are contraindicated for a large percentage of the population. Safer, yet still effective inactivated and subunit vaccines are needed, and epitope identification is an essential step in the development of these subunit vaccines. In this study we focused on 4 vaccinia membrane proteins known to be targeted by humoral responses in vaccinees. In spite of the narrow focus of the study we identified 36T cell epitopes, and provide additional support for the physical linkage between T and B epitopes. This information may prove useful in peptide and protein-based subunit vaccine development as well as in the study of CD4 responses to poxviruses.

Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers.

Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300-500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor-intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host-cell DNA levels for human vaccines. This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine sulfate and Toyopearl AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was approximately 60% for both pseudo-affinity MA and the Cellufine sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo-affinity chromatography materials. Cellufine sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo-affinity with anion exchange MA a further reduction of host-cell DNA was achieved.

Immunogenic properties of plant-derived recombinant smallpox vaccine candidate pB5.

The extracellular virion membrane protein B5 is a potent inducer of immune responses capable of protecting mice and primates against poxvirus infections. Here, we examined the antibody response induced in mice immunized intramuscularly (i.m.) or intranasally (i.n.) with plant-derived B5 (pB5) accompanied or not with plant total soluble protein (TSP) at various concentrations. Increasing amounts of TSP inhibited the pB5-specific response in both i.m.- and i.n.-immunized mice, with more dramatic effects in the latter. pB5 administered to mucosal surfaces induced specific IgG and IgA responses, whereas i.m. immunization produced high serum IgG titers and no IgA. A 6-fold increase in pB5 dosage administered i.n. led to an antibody response comparable to that obtained by i.m. injection. Our study addresses the quality/quantity issues of the pB5 subunit preparation and demonstrates the feasibility of mucosal administration of plant-derived smallpox subunit vaccine in obtaining a potent immune response. Overall, this work points to the practicability of needle-free mucosal administration of such vaccines in light of purity, dosage and adjuvant formulation.

The US smallpox vaccination program: a review of a large modern era smallpox vaccination implementation program.

We describe the US experience with a large-scale smallpox vaccination program in the modern era and quantify the anticipated and unanticipated local and systemic side-effects of smallpox vaccination. In addition, we review unexpected issues, such as the development of myopericarditis discovered during the implementation of this program. These results constitute the largest dataset of a vaccinia vaccination program utilizing calf-lymph derived New York City Board of Health strain vaccine (Dryvax, Wyeth) since the 1970s. These results should inform current and future vaccinia vaccination programs and provide a historical rate of complications against which candidate vaccine side-effects can be compared in future clinical trials.

[Jenner's cowpox vaccine in light of current vaccinology]. / Jenners koepokentstof in het licht van de moderne vaccinologie.

Two hundred years ago Edward Jenner inoculated James Phipps with vaccinia and 181 years later smallpox had disappeared from the surface of the earth as a result of generalized vaccination. Compared to the requirements of modern vaccinology, the procedures used by Jenner and his successors, were extremely primitive because of an almost total lack of knowledge in the field of microbiology and immunology. The active principle of smallpox vaccine is vaccinia virus, which in many respects, differs from that of natural cowpox; the term "cowpox" has been used for more than a century and a half to designate the vaccine; it appears itself to be a misnomer, because it is most probably by a virus of rodents, which only occasionally infects bovines or other species, especially cats. The origin of vaccinia remains doubtful, but a plausible explanation is that it is derived from horse-pox. Jenner was convinced that he was working with a virus of equine origin, which was occasionally transmitted from the horse to the cow by the personnel on the farms. Horse pox has now completely disappeared. Especially during the first years after Jenner's discovery, great confusion was caused by other lesions on the cow's udder, which were called "spurious cowpox". We know today that these lesions could be caused by the viruses of papular stomatitis, pseudo-cowpox or para-vaccinia (milker's nodules), herpes mammilitis and papillomatosis; they could not be differentiated from those of cowpox or vaccinia, in addition lesions due to bacteria or other causes also led to confusion. During the first eighty years the vaccine was being transferred almost exclusively from arm to arm with the risks inherent in this procedure; one of the reasons for applying this method was the fear of "bestialization" thought to be linked with the use of material of animal origin. Several contaminations have been observed as a result of the use of the arm-to-arm procedure: smallpox was transmitted, especially in the beginning, because vaccinations were carried out in a contaminated environment. Syphilis was diagnosed in several countries after the use of vaccine taken from syphilis patients. At least two foci of hepatitis were reported after the use of contaminated human lymph. Transmission of tuberculosis or what was then designated as scrofulosis was unlikely, but was used as one of the main arguments against vaccination by the antivaccinists. Varicella and measles were transmitted from time to time with the vaccine and also bacterial infections, such as staphylococci, streptococci e.a. From the global point of view, however, the number of contaminations remained limited in comparison with the large numbers of vaccinations that were performed. Another problem the early vaccinators were facing, was that of the decline and disappearance of the immunity after a certain number of years. Jenner and his successors believed that the immunity post vaccination would be lifelong as it was after variolation. When in the early part of the 19th century more and more immunity breakdowns occurred, this observation led to total confusion and it took dozens of years of debate and controversy before the only logical and efficacious measure, i.e. revaccination, was generally accepted and implemented. In the last third of the 19th century "human lymph", obtained by arm-to-arm vaccination, was gradually replaced everywhere by animal lymph i.e. vaccine produced on the skin of animals, mainly calves. The determining factor in the switch was the risk of vaccination syphilis. Everywhere vaccine institutes were created, where the vaccinia virus was propagated on the skin of calves. The harvested virus served each time for the inoculation of fresh calves; this resulted in a gradual increase of the number of passages leading to the possible risk of overattenuation. To avoid this risk, passages in man, donkeys, rabbits or other species were performed from time to time.

The origin of HIV-1, the AIDS virus.

This article proposes a series of experiments to determine if cows and sheep could be used as animal models for HIV-1, the AIDS virus. To justify this effort, a substantial case is presented that HIV-1 is a natural recombinant of Bovine Leukemia Virus (BLV) and Visna Virus. This natural recombinant may have been inadvertently transferred to humans through the Intensified Smallpox Eradication Program conducted in sub-Saharan Africa in the late 1960s and most of the 1970s.

PIP: HIV-1 is a natural recombinant of Bovine Leukemia Virus (BLV) and Visna Virus. The author posits that this recombinant virus may have been transferred to humans through the Intensified Smallpox Eradication Program conducted in sub-Sahara Africa in the late 1960s and most of the 1970s. If this is the case, knowledge thereof could help in the search for a cure or vaccine against AIDS. The paper discusses the correlation between the distribution of AIDS cases worldwide and the vaccination program which eradicated smallpox; offers facts about BLV, Visna, HIV-1, and the recombination of retrovirus genomes; and describes a series of experiments to determine if cows and sheep could be used as animal models for research into HIV-1. Weak points of the theory are also presented, such as why AIDS has appeared only recently even though the same method of vaccinating against smallpox has been used for almost 200 years.

Cutaneous responses to smallpox revaccination with calf lymph and the effect of fluorocarbon purification of the vaccine.

For several years the routine smallpox revaccination procedures at a central inoculation unit were arranged to assess the relation between vaccine titre (pock count) and success. Calf lymph batches were applied, diluted and undiluted, over a log titre range of 7.0-9.8. The dose-effect relationship did not appear to fit a linear equation on a log-probit scale, except in the lower part of the titre range. Plotted on this scale, the take rates of nearly all vaccine specimens of the required strength (> 10(8) PFU/ml) were lower than anticipated by linear extrapolation from low titres. Differences between batches were noted. These findings relate to pulp processing without purification. Fluorocarbon extraction of the calf skin harvest resulted in a 4-fold increase of vaccine volume with commensurate virus dilution; it also gave clearly higher take rates than parallel nonpurified vaccine specimens, whether at original strength or at 4-fold dilution.

Virus particle content of smallpox vaccines.

Calf lymph smallpox vaccines contain too much extraneous debris for an accurate assessment of their virus particle content. The process of partial purification of the vaccine utilizing enzymatic digestion by chymotrypsin, subtilisin, and collagenase solubilized enough debris to permit electron microscopic virus particle count. Enzyme treatment did not degrade or destroy the virus nor did it reduce the infective titer. Commercial vaccines studied ranged in virus content from 1.89 x 10(9) to 1.09 x 10(11) virus particles/ml. The pocking efficiencies on the chorioallantoic membrane of some of these vaccines varied from 200 to 1,200 virus particles per pock-forming unit.