There are several reasons to justify a replication-competent, “live”, vaccine as an attractive approach to control LF: (i) cell-mediated immunity plays the major role in LF patient recovery and in protection; (ii) a live vaccine provides the most effective natural pathway to process and present protective antigens to MHC molecules; (iii) epidemiological observations provide evidence that a single LASV exposure will induce long-term protection against disease [10
]; and (iv) a vaccine candidate formulated to contain both LASV NP and GP antigens will induce a broad cross-reactivity and strong CD4+ memory T cells against all phylogenetic groups of LASV [19
Safety experiments in common marmosets confirmed our previous data in rhesus macaques [19
]. Notably, the ML29 reassortant was detectable in plasma by conventional plaque assay only in one sample after immunization at high dose (). Low-dose immunization resulted in transient ML29 replication in tissues at very low levels. High-dose immunization resulted in more extensive virus replication. Still, this replication was transient, moderate (2–4 log10
PFU/g), and well controlled. However, ML29 immunization at high dose can damage the blood–brain barrier in some marmosets.
Immunization at low dose did not result in detectable virus shedding, whereas transient viral shedding was detected in one marmoset, CJ12, immunized at high dose (). Although shedding by the respiratory route after immunization at high dose cannot be excluded, transmission of vaccine virus by this route is very unlikely. Entry of arenaviruses occurred only via basolateral receptors [32
] suggesting that ML29 can infect airway epithelia only when epithelial integrity is compromised.
To further characterize ML29 safety features we used transcriptome profiling in human PBMC. Exposure of PBMC samples to ML29 and MOPV showed changes in 400 genes but only 14 displayed opposite regulation. Interestingly, MHC-II DR beta 5 gene was significantly up-regulated in MOPV-exposed vs. ML29-exposed cells. However, immunochemistry analysis () showed strong up-regulation of MHC-II class genes at the protein level suggesting that the transcriptional and translational status of the various HLA-DR loci can vary in MOPV- and ML29-exposed cells. Overall, as seen in , the ML29 gene expression pattern in human PBMC was closer to the pattern of mock-exposed cells and clearly maintained a significant distance from the MOPV-exposed group.
Immunization of marmosets with ML29 increased populations of CD14+ cells and CD3+ T lymphocytes in circulating blood. We also saw recruitment of CD3+ T cells and over-expression of HLA-DR, P, and Q in target tissues (). Taken together these data indicate that ML29 immunization resulted in antigen stimulation. In contrast, LASV infection in marmosets was associated with lymphoid depletion, marked reduction in CD3+, CD20+ cells, and down-regulation of class II MHC antigens [22
Our results in rhesus macaques [19
] and in marmosets (present study) indicate that a single s.c. inoculation of ML29 vaccine at low dose induces specific cell-mediated T cell responses assayed in IFN-γ or TNF-α ELISPOT. These responses seem to be responsible for complete protection against fatal LF disease. In confirmation of these observations target tissues of vaccinated and challenged animals collected at the end of the experiment had no histological alterations and, notably, were free from infectious LASV (Tables and ). In ML29-immunized primates we detected very weak antibody responses measured by IgG ELISA (, see also Ref. [19
The existence of natural reassortment raises the concern that vaccination with an attenuated reassortant vaccine in the presence of the virulent strain will somehow accelerate disease spread. However, experiments with Rift Valley fever virus showed that reassortants between the wild-type virus and a live attenuated ML-12 vaccine generated only phenotypes with protective activity against the disease [33
]. Studies on molecular epidemiology of infectious bursal disease virus (IBDV) confirmed the existence of natural reassortants between vaccine and wild-type IBDV strains. However, the reassortant that exhibited a segment from a natural virulent strain and a segment from a vaccine strain induced significantly less mortality than typical wild-type IBDV [34
]. No evidence of wild-type reversion has been observed in recovered FluMist vaccine strains that have been tested. In clinical trials transmission of FluMist virus has been documented only in a single person, who remained asymptomatic [35
Natural reassortment between arenaviruses has not been described so far. Recently we have performed simultaneous vaccination-challenged experiments in guinea pigs and showed that simultaneous application of ML29 and LASV attenuates wild-type infection and protects animals [21
]. All these data indicate that genetic reassortment with wild-type viruses during a vaccination process would also be expected to yield attenuated variants and to reduce the overall incidence of disease.
In summary, a ML29 reassortant is safe, immunogenic, and induces complete protection at low dose against LF in non-human primates. Based on cross-protective studies in guinea pigs [21
], it is reasonable to expect that ML29 vaccination will also protect non-human primates against phylogenetically diverse LASV isolates. Cell-mediated immunity seems to be responsible for effective LASV clearance from blood and tissues of immunized animals [19
]. ML29 phenotype and genotype have been stable for at least 12 passages in tissue cultures. None of the existing LF vaccine candidates [9
] can share these ML29 features. The recent demise of two medical doctors in Nigeria [39
] is one more reminder that an effective vaccine for LF is urgently needed and that local health workers should be the first people offered this vaccine. In West Africa as many as a tenth of the potential vaccinees have an altered immune status. For these individuals the risk of adverse effects should be carefully evaluated and compare with vaccination benefits. To address this issue, experimental efforts are under way to determine whether ML29 is still safe and effective in the monkey model for AIDS.