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In the middle of the 18th century, inoculation against smallpox became more and more common, and attempts were also made to test the same principle, viz. inoculation with the agents causing the disease for other human and animal diseases. It was tried for rinderpest, measles and sheep pox. In addition, there were some suggestions for using the principle against the plague. The disease had disappeared from Western Europe by this time, but still raged in eastern countries, such as Russia. However, the government rejected the proposal for trial inoculations in Moscow. During the first half of the 19th century, the plague was still widespread in the Middle East, where different European doctors worked on combatting it. The first documented inoculation trial was carried out by a certain Mr. Whyte, an English physician who inoculated himself and four assistants in 1801. All five died a few days later. In the following years, more tests were carried out, inter alia: in 1802, by Desgenettes, the chief physician of the French army in the Middle East; in 1803, by Eusebio Valli, an Italian physician in Constantinople; in 1818 and 1819 by Sola, a Spanish physician in Tangier. However, none of these tests produced clear results. During the epidemic in Egypt in the 1830s, further inoculation tests were carried out by a group of French plague specialists with the main aim of establishing whether the plague could be transmitted between humans. These tests did not result in any clear conclusions either. Following the discovery of the plague bacillus at the end of the 19th century, a number of different live and dead vaccines were developed, and were also used in endemic areas, but the level of efficiency has never become very clear. This is not really surprising, as even the disease itself often does not provide strong immunity, and reinfections are by no means uncommon.

Louis Willems's name is intimately linked with the history of prophylactic immunization in the nineteenth century. When he obtained his medical degree in 1849 contagious bovine pleuropneumonia or lung sickness was raging among the cattle population in most European countries. As the son of a cattle fattener Willems was confronted directly with the problem in his father's stables and decided to study the disease and to search for a remedy to combat it. The disease is caused by Mycoplasma mycoides and subspecies mycoides, but in the middle of the nineteenth century during the battle between the miasmatists and the contagionists, many had doubts about its contagiousness. Willems defended from the start the contagiousness of the disease and noticed that animals who had survived an infection did not contract it a second time. He demonstrated that inoculation of the serous fluid from the lungs or from the pleural cavity of affected animals into healthy cattle led to pronounced local reactions. When these inoculated animals later on came into contact with diseased cattle they were shown to be immune. In his first trials he inoculated at the base of the tail or around the nostrils but this led to very severe reactions and frequently to death. He then started inoculating at the tip of the tail with much better results. Most animals showed a more or less pronounced reaction at the inoculation site and about seven percent lost their tail partially or completely through necrosis, but the mortality remained very limited. The local reactions were caused by the etiological agent itself. The lesions in the connective tissue of the tail showed much resemblance to those in the interlobular septa of the lungs and contained strong accumulations of serous fluid. The tip of the tail was obviously a good choice; this was confirmed later by many authors and the procedure is still being used today in areas where the disease is still prevalent. Inoculation at other sites of the body such as the neck or the dewlap, led to very severe reactions often followed by death. Willems also demonstrated that local inoculation at the tip of the tail not only immunized the animals against infection via the respiratory tract resulting from contact with diseased animals, but also against a second inoculation in the tail, in the neck or elsewhere. Material harvested from the inoculation site in the tail (so-called secondary "virus") could also be used as inoculum. Animals who showed no reaction to the first inoculation received a second one after a few weeks. Immunization as a result of inoculation was proved repeatedly experimentally as well in Willem's lifetime, by himself and by his contemporaries, as later in more recent trials. Failures were usually attributable to inoculation of already infected animals or to the use of badly stored or purulent inocula. Inoculation during the incubation period did not provide protection. Willems' concepts about the mechanisms of immunity were understandably vague and ill-defined. He considered pleuropneumonia as an affection of the whole body with process in the lung in case of natural infection; following inoculation this process took place somewhere else and one created as it were a typical lung infection at another site of the body. Through the introduction of the "virus" a "dynamisation" of the whole body took place by which the blood and other organs became insensitive to reinfection. This explained why the inoculation protected not only the inoculation site, but the whole organism; Willems thought that the infection did not spread from inoculated to non-inoculated animals; this opinion was supported by some other workers in the field but opposed by others. The publication of his results created enormous interest in his country and abroad. In several countries commissions were created, trials were initiated and several foreign observers came to visit Willems in Hasselt. In general his results were confirmed abroad at least

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.

A strain of human cytomegalovirus called Towne was isolated in WI-38 human fibrolast cell cultures from the urine of an infected infant. It was then passaged 125 times in WI-38, including three clonings, and a pool was prepared in the same cell substrate for use as a potential live attenuated vaccine. The Towne virus has a broad antigenicity and cross-reacts with the AD-169 strain. Several markers of the Towne virus were found which differentiated it from fresh isolates. One of these was resistance of the former to trypsin. The Towne virus was tested for freedom from oncogenicity or other harmful effects in preparation for tests in humans.

The Alice strain of live attenuated influenza virus was obtained by selection of a gamma inhibitor-resistant strain from a virus recombinant between A/PR/8/34 (HON1) and A/England/42/72 (H3N2). Its behaviour in vitro and in vivo was studied. Three marker systems were investigated: resistance to serum inhibitors, growth capacity at high temperature and low sensitivity to amantadine hydrochloride. In ferrets the strain was found to be attenuated and immunogenic. Passages in man, animals and eggs have not affected its resistance to gamma inhibitors.

Human fibroblasts are the only cells that regularly produce cytomegalovirus of adapted laboratory strains and release fair amounts of cell free virus. One such strain, Towne, was adapted to WI-38 and could release 10(6) to 10(7) p.f.u. per ml tissue culture fluid. The maximum cell-free virus is released at 5-7 days after infection, when cytopathic effect has reached a peak. The virus harvest must be combined with stabilizer rapidly and rapidly lyophilized or frozen, for the virus is unstable even at 4 degrees C. With regard to control, cytomegalovirus is poorly neutralized by antibody-containing serum, and complement is required for maxium neutralization. Frequent addition of fresh antiserum may be necessary to keep cytomegalovirus from replicating in cells inoculated to detect other viruses. Another special problem is the putative association of oncogenicity with herpes group viruses. Tests must be done in newborn hamsters to show that live or inactivated virus does not cause tumors. Towne vaccine tested in man has produced asymptomatic infection with antibody response. No urine excretion has been observed, and antibodies persist for at least 2 years.

In 1967-1968 a trial of "Cendehill strain" rubella vaccine was initiated in a group of 3 to 22 year-old prescreened seronegative girls, most being between 8 and 16 years of age. Sera from the vaccinees were taken at regular intervals during a period of 8 to 10 years. The evolution of the antibodies was followed over this period. No antibody titer change was observed in 75 to 78% of the sera. A four fold increase was shown in 6 to 10% and a four fold decrease in 15% of the vaccinees. All sera still showed a positive titer at the end of the period. The importance of these results for the final objective of rubella vaccination will be discussed.

Triple seronegative calves were exposed by the nasal route to three (ts) mutants of bovine respiratory viruses (PI3, IBR, Adeno3). After a single exposure, they responded with significant levels of serum antibodies to the three viruses. Nasal antibodies were demonstrated for PI3 and adenovirus antigens. The failure to demonstrate nasal antibodies to IBR may be due to lack of sensitivity of the procedure used. When reexposed six weeks later, calves had sharp increases in levels of serum antibodies and developed a secondary type response at the local level for all three viruses. The persistence of the local antibodies was much longer after reexposure than after primary inoculation. This study indicates that the simultaneous application of these three (ts) viruses by the respiratory route is perfectly safe and affords a long lasting immunity towards homologous respiratory infections.

The persistence of systemic and local antibodies was studied after two intranasal administrations of the vaccine, six weeks apart. Systemic antibodies to I.B.R. and adenovirus 3 evoked by the vaccine were still present 21 weeks following the second dose of the vaccine. Inconslusive results were obtained regarding the persistence of systemic and local PI-3 antibodies because of an intercurrent natureal PI-3 infection occurring during the observation period. Local antibodies to adenovirus type 3 were found in a high percentage of vaccinated animals 21 weeks after the second dose of the vaccine, whereas local antibodies to I.B.R. remained detectable in 50% of the animals eight weeks after thesecond dose. The results of a challenge study 21 weeks after revaccination show that the presence of local and systemic antibodies prevent the multiplication of PI-3 and BAV-3 in the upper respiratory tract. Protection against I.B.R. was achieved in the absence of detectable local antibodies.

Recently, new methods for the selection of candidate live influenza vaccine viruses have become available. They are based on the characterization of the genetic material of these viruses and have been shown to correlate with attenuation for man. Adequately attenuated viruses have been obtained from various H3N2 strains using standard methods of attenuation for each strain. These vaccine strains met the criteria for live attenuated influenza virus vaccines: they were attenuated, non-transmissible and immunogenic. The use of modern methods of attenuation also allows rapid updating of live influenza virus vaccines. Laboratory methods used, the safety testing of candidate vaccines in animals and man will be discussed. There is an urgent need for standardization and for the establishment of minimum requirements and specifications.

Ligated intestinal loops of mice were found suitable for the assay of heat-stable enterotoxin produced by enteropathogenic Escherichia coli strains of porcine origin; loops inoculated with heat-labile enterotoxin failed to react.

The multiplication pattern of a temperature-sensitive (ts) mutant of bovine parainfluenza 3 virus was studied in hamsters and compared with that of a virulent virus strain. The ts mutant was recovered regularly from the nasal mucosa and not from the lungs, whereas the virulent virus multiplied in the lungs as well as in the nasal mucosa.The serological response induced by the mutant was comparable to that obtained after inoculation of the virulent virus.This ts mutant may be a potential candidate for a live intranasal vaccine against bovine parainfluenza 3 infection.

A live attenuated influenza virus ("Ann" strain) derived from A/England/878/69 was given intranasally to a group of volunteers, most of whom had already circulating antibodies against H(3)N(2) viruses at the time of inoculation. There was a fourfold or higher increase of circulating hemagglutination-inhibiting antibodies in those volunteers who had relatively low initial titers. The response was lower in those with initially higher serum titers. The pattern of the serum neutralizing antibody response was very similar. The geometric means of the antineuraminidase antibodies were 67 and 118 pre- and postvaccination, respectively. All subjects showed a rise in local neutralizing antibodies in their nasal secretions with geometric means of 4 and 17 pre- and postvaccination, respectively. The levels of local antineuraminidase antibodies also rose in most subjects. In addition to the response to the homologous virus type, the antibody formation to the recent A/England/42/72 was measured in the sera and nasal secretions of some subjects. There was a clearcut response in most of the sera and in all of the secretions examined. The stimulation of circulating lymphocytes was measured in 6 volunteers. All volunteers showed a temporary stimulation. The stimulation index ranged between 2.5 and 28.5.