The vaccine was well tolerated, and there were no adverse reactions, neither swelling after the injections nor systemic reactions. During the earlier experiments with this vaccine in Iceland the experience was the same .
The antibody response to this vaccine was previously studied in seronegative Icelandic sheep kept in isolation, in spite of the fact that Iceland has been free of Maedi-Visna infection since 1965 . In this field trial we did not test for antibody response after vaccination, because the vaccinees were exposed to natural infection from their environment every day after their birth, and therefore it was impossible to be certain whether they were responding to the vaccination or early natural infection from their environment. For the immune response to the vaccine we therefore refer to the studies done in isolation of the 16 seronegative Icelandic vaccinees, mentioned here in the Background section .
As mentioned here in the Background section, a small experiment on 5 twin-pairs, one twin in each pair vaccinated and the other an unvaccinated control, showed, that after 4 years of close contact with sheep infected with the vaccine strain of virus all the unvaccinated twins had seroconverted, but only 2 of their vaccinated siblings. Cultivation of virus from lungs, spleens, hilus lymph nodes and choroid plexuses from all 10 twins confirmed these results. The 3 seronegative twins tested were also negative in all the tissue culture experiments . This type of work can only be done at laboratory conditions, not at the natural conditions on an infected farm. In slow infections were you count the incubation period in years without clinical signs, you have to select the indirect method of antibody response as the most reliable method to separate real infection from contamination of specimens taken at field work. In this field trial the results shown in Table 2 for Group 1 at 28 months remind of the results found in the earlier twin experiment in Iceland and may mean protection, although our methods did not directly prove it. Therefore we prefer to call our findings in this field trial a delay of infection in the vaccinees which responded to the vaccination.
Methods to detect infection in vaccinees and their unvaccinated controls
Those who are familiar with the diagnostic work on the HIV lentivirus infection in humans know, that positive ELISA test is not a reliable diagnostic test for HIV infection and must be confirmed by a more reliable test, e.g. Western Blot (WB) test on the ELISA positive serum. In most of these cases the ELISA test is false positive. On these grounds we selected another and more reliable test for this field trial.
In the HIV laboratory of the Department of Microbiology, University of Iceland, we developed Western Blot technique for Maedi-Visna and compared it with the CF test we had previously developed and used extensively during the eradication of the disease from Iceland [7, 8]. The comparison with the WB test showed that the CF test became positive at the same time as the WB in all cases tested . Therefore, the CF test, simple and reliable in diagnostic work on many human virus diseases became the test for natural infection in this field trial instead of the cumbersome Western Blot technique. If you compare the vaccinees with their unvaccinated controls as shown in Groups 1 and 2 in Table 2, you see that both the unvaccinated control twins and the vaccinated twins that did not respond to the vaccination became positive in the CF test at the same period of time. If you look at Group 1 only, you see that it is during the 2nd year of the experiment that they develop antibodies if they are infected from the outside environment, but earlier if the infection is milk-born as in Group 2 in Table 2. If you look at Group 1 in Table 2 early in the 3rd year of the experiment you see that only 2 unvaccinated sheep in the control group are seronegative, but 9 of their vaccinated siblings are still seronegative 2 years after their vaccination program ended.
Boosters of vaccine
In this experiment no “boosters” of vaccine were given to vaccinees after 3 months of age. Infections of lambs in Group 3 are apparently not milk born infections, although their mothers were seropositive both in AGID and WB tests. This is in contrast to the experience among unvaccinated twins born by seroconverting mothers in Group 2. The lambs in Group 3 might have benefited from vaccination after 6 months of age, when passive maternal antibodies should no longer be interfering with the vaccination. Boosters late in the first year might also have prolonged the period of delay in those vaccineees in Groups 1 and 2, which responded to the vaccination. Unsuccessful vaccination in Group 3 is most likely caused by interference of passive maternal antibodies with vaccine given so early in life, a common finding if children are vaccinated during their first months of life.
Clinical maedi-visna disease
The infected sheep flock, in which this experiment was conducted, was heavily contaminated with Maedi-Visna virus as judged by the tests on the mothers of the vaccinees and the rate and time of infection of the unvaccinated twins in Groups 1 and 2. Yet, there was no clinical illness in this flock and the sheep thrived well, although they were infected. Clinical Maedi-Visna is very rare in Cyprus, although the flocks are infected. The Icelandic experience from the importation of Maedi-Visna in 1933 until its total eradication in 1965, was different. There, clinical Maedi-Visna was disastrous in good sheep farming areas, culminating in the eradication by slaughter of all the infected flocks .
The virus strains isolated from infected sheep in the flock housing this experiment were very slowly growing and it took many passages in tissue culture to obtain working titers of these strains. In spite of the obvious strain difference between the circulating virus strains isolated from the flock and the old Icelandic vaccine strain K796 (Table 1), monovalent vaccine made by inactivation of whole virus K796 apparently delayed infection in more than half of the number of vaccinees in Groups 1 and 2, when compared with their unvaccinated siblings living in the same infected environment and suckling the same mothers.
Vaccine preparation and methods of challenge
More than twenty years ago two groups of workers, a multinational Scandinavian group and an American group, reported unsuccessful attempts at vaccinating sheep with inactivated Maedi-Visna and ovine progressive pneumonia (OPP) vaccines [10–13]. Both groups used methods very different from those used in the work described here. They used different vaccine strains of virus, different methods of virus inactivation and concentration, different adjuvants, if they were used, and last but not least different methods to challenge their results. Both groups used high-titer live virus to test the results of their vaccination. The Scandinavian group gave to their vaccinees and controls live virus by the respiratory route, but the American group injected large amounts of high-titer, live virus intravenously. Neither group had found neutralizing antibodies in their vaccinees, in contrast with the previous Icelandic experience with this vaccine . The question arises as to whether it will ever be possible to determine if a lentivirus vaccine is working, by inoculating high titer of live virus directly in to the circulation of the vaccinee, where susceptible living cells, such as macrophages or T-cells, are located and may become infected. Such cells may carry the virus to distant organs, such as lymph nodes and lungs, where it can further multiply. Those who work with lentiviruses need to develop more natural challenge methods, although it takes time. Therefore, it needs to be remembered that a challenge by injecting a high titer of live virus is not comparable with the natural routes in an infected sheep flock.
Through 19 years of work with this vaccine, all attempts to grow virus from concentrates of vaccine following inactivation of virus in tissue culture fluid have turned out negative. This may be relevant to those who have problems with other lentivirus vaccines and are against the idea of using whole inactivated virus in their experiments. It is, apparently, possible to treat a lentivirus by those conventional methods, that are used to produce good inactivated vaccines against poliovirus, hepatitis-A virus and influenza virus.