An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause arenavirus disease but does elicit Lassa virus-specific immunity

Abstract

Background: Lassa hemorrhagic fever (LHF) is a rodent-borne viral disease that can be fatal for human beings. In this study, an attenuated Lassa vaccine candidate, ML29, was tested in SIV-infected rhesus macaques for its ability to elicit immune responses without instigating signs pathognomonic for arenavirus disease. ML29 is a reassortant between Lassa and Mopeia viruses that causes a transient infection in non-human primates and confers sterilizing protection from lethal Lassa viral challenge. However, since the LHF endemic area of West Africa also has high HIV seroprevalence, it is important to determine whether vaccination could be safe in the context of HIV infection.

Results: SIV-infected and uninfected rhesus macaques were vaccinated with the ML29 virus and monitored for specific humoral and cellular immune responses, as well as for classical and non-classical signs of arenavirus disease. Classical disease signs included viremia, rash, respiratory distress, malaise, high liver enzyme levels, and virus invasion of the central nervous system. Non-classical signs, derived from profiling the blood transcriptome of virulent and non-virulent arenavirus infections, included increased expression of interferon-stimulated genes (ISG) and decreased expression of COX2, IL-1β, coagulation intermediates and nuclear receptors needed for stress signaling. All vaccinated monkeys showed ML29-specific antibody responses and ML29-specific cell-mediated immunity.

Conclusion: SIV-infected and uninfected rhesus macaques responded similarly to ML29 vaccination, and none developed chronic arenavirus infection. Importantly, none of the macaques developed signs, classical or non-classical, of arenavirus disease.

Figure 1

Experimental design. Sixteen rhesus macaques were inoculated with SIVmac251 and monitored for viral loads and SIV-specific immunity. Five succumbed within a year after SIV inoculation (48, 222, 223, 231, and 240 days after SIV infection) and the remaining 11 monkeys were vaccinated with ML29 or LCMV-Armstrong on day 393 after SIV-infection. A month after the vaccination, five monkeys (ML-1, SIV/ML-1, SIV/ML-2, SIV/ML-6, SIV/ML-7) were boosted with ML29. Necropsies for 6 animals were performed on days 426, 452, 458, 491, 505, and 516 (after SIV). Five months after ML29 vaccination, 5 animals were still surviving. (X) indicates the relative position on the timeline of samplings and necropsies.

Figure 2

Plasma SIVmac251 viral loads after infection. Sixteen monkeys were classified based on the viral load peak after SIV infection as follow: (A) high, (B) medium, or (C) low. Viral loads were monitored up to 2 years after SIV infection. 7 months after (represented as a space in the X axis) eleven surviving monkeys were vaccinated with ML29 and SIV viral loads were reported up to 7 weeks. * denotes monkeys that died before ML29 vaccination. D) Percentage of variation in SIV viral loads is depicted over the first 3 weeks after ML29 vaccination. Seven of the 8 SIV/ML29 monkeys experienced a drop in SIV (median 20%) during the first week after vaccination, but those titers returned the following week.

Figure 3

Survival of macaques after SIV-infection and ML29 vaccination. Eleven of 16 rhesus macaques that had been infected with SIVmac survived to be enrolled in a ML29 vaccination study (day 393 after SIV infection). Macaques were classified as rapid (>10⁶ SIV RNA mol/ml at setpoint), median (10⁴ – 10⁶ RNA mol/ml), and slow (<10⁴ RNA mol/ml) AIDS progressors. Eight were vaccinated with ML29 and three were given LCMV-Armstrong, and all were monitored for signs of arenavirus disease. Five months after vaccination, 5 healthy animals remained, having survived 530 days after SIV infection. The absence of gross signs of arenavirus disease and the absence of clinical signs in blood and tissues samples indicated that vaccinated animals were protected from arenavirus disease.

Figure 4

Detection of arenavirus in ML29-inoculated macaques. Virus titration by plaque assay revealed that only three animals (SIV/ML-1 •, SIV/ML-3▴, and SIV/ML-8 ▵) showed low viremia by week 3 after inoculation with ML29. Negative samples were tested by a second round of amplification (see Methods), showing traces of virus in two animals (ML-3 × and ML-4 ⊙), the remaining animals (▪) tested negative until the end of the study.

Figure 5

Percentage of monocytes and NK cells in PBMC after ML29 vaccination. A) In the 8 SIV-infected, ML29-vaccinated monkeys there is a significant reduction of the percentage of circulating NK (CD16+) cells one week after vaccination. B) Reduction of the NK (CD16+) population in four healthy (non-SIV-infected) animals one week after ML29 vaccination. C) In the 11 SIV-infected, arenavirus-vaccinated monkeys there is a slight increase in monocytes a week after ML29 vaccination. D) This increase is also observed in non-SIV-infected control monkeys.

Figure 6

Gene expression of SIV-infected monkeys given the ML29 Lassa vaccine. Column one has the Genbank gene name. Column two has the fold difference in gene expression between ML29-vaccinated and uninfected (no SIV) monkeys, week 1. Column three has the fold difference in gene expression between ML29-vaccinated and SIV-infected, week 1. Column four has week one gene expression data from our previous profiling of monkeys inoculated with virulent and non-virulent strains of LCMV [35]. The blue star indicates indicates potential virulence markers in primate and guinea pig infections with virulent/mild virus pairs.