Exploring of primate models of tick-borne flaviviruses infection for evaluation of vaccines and drugs efficacy.

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8-16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops

Viral titers (log₁₀PFU/ml) in CNS and visceral organs of <i>C. aethiops</i> monkeys, immunized twice with TBE inactivated vaccine (FSME-Immun), and in non-immunized monkeys on the 23–26 days after s/c challenge with 6.7 log₁₀ PFU of TBEV, strain Absettarov.

<p>0–<1 PFU in 0.1 ml of 10% suspension.</p

Virus titers (log₁₀ PFU/ml) and the presence or absence of viral RNA (+/−) in CNS and visceral organs of <i>C. aethiops</i> monkeys immunized and non-immunized with inactivated vaccine against TBE (EIPVE) on the 14–16 days after challenge with 7.3 log₁₀ PFU of OHFV, strain Nikitina.

<p>0–<1 PFU in 0.4 ml 10% suspension.</p><p>“−”– RT-PCR is negative for viral RNA.</p><p>“+”– RT-PCR is positive for viral RNA.</p

Virus titers in visceral organs and CNS (log₁₀ PFU/ml) of <i>M. fascicularis</i> monkeys s/c inoculated with TBEV Abs-18 or Sof-16 strains at the early terms after infection (8–10 d.p.i.).

<p>0–<1 PFU in 0.2 ml of 10% suspension; s.c. – spinal cord, l.n. – lymph nodes.</p

Histological lesions in the brain of monkeys infected with 6.0 log₁₀ PFU of Abs-18 at the late terms after infection (27–28 days).

<p>A and B. Brainstem (<i>truncus cerebri</i>) of <i>M. fascicularis</i> monkey #26: multiple small vasculitis (1), perivascular edema (2), degenerative changes in neurons (3). Magnification: (A) ×100, (B) ×200. C and D. Subcortical region of <i>M. fascicularis</i> monkey #26: small vasculitis (1) and nodules of neuronophagia (2). Magnification: (C) ×100, (D) ×400. Cortex of cerebellum: E. Fall out of small groups of Purkinje cells in <i>M. fascicularis</i> monkey #34 (1). Magnification ×100.; F. Non-infected normal control. Magnification ×200. Staining by Nissle method was used.</p

Histological lesions in the liver and spleen of <i>M. fascicularis</i> monkey (#26) at the late terms after infection with 6.0 log₁₀ PFU of Abs-18.

<p>A. Liver: lymphohistiocytic infiltration of portal liver tracts (1). Magnification ×200. B. Spleen: reduction of lymphoid follicles (1); depletion of white pulp along the trabecular arteries (2). Magnification ×400. Staining with hematoxylin and eosin was used.</p

The complete blood count in <i>C. aethiops</i> monkeys before and at the different terms after s/c challenge with 7.3 log₁₀ PFU of OHFV strain Nikitina.

<p>ND – not determined; aspartate aminotransferase (AST); alanine aminotransferase (ALT).</p

Study design.

<p>The flow chart represents the study design and performance. The monkey species used in each experiment is shown in red. Vaccine: the name of the vaccine administered before challenge or “no” vaccine is indicated. ^a - the number of monkeys used in each experiment is shown in parenthesis. ^b - the blue framed box represents the section number, which describes the results of each particular experiment. The challenging virus is shown in blue. “Analysis made”: the types of analysis and parameters used to evaluate the model are listed. “Results0 are presented in”: the results presented in figures or tables in the current study are listed.</p

Viremia and neutralizing antibody titers in monkeys infected with 7.3 log₁₀ PFU of OHFV.

<p>A. Viremia in two non-vaccinated monkeys (#1 and 3). B. Viremia in two monkeys (# 2 and 4), vaccinated with commercial inactivated vaccine against TBE (EIPVE). Viremia was analyzed in blood clots by plaque assay; the limit of detection of the plaque assay was 0.5 log₁₀ PFU/ml. D.p.i. – days post-infection. C. Average (for two monkeys) reciprocal neutralizing antibody (NA) titers in sera of immunized and non-immunized <i>C. aethiops</i> monkeys before and after challenge. Two different viruses were used for neutralization assay: TBEV or OHFV.</p