Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses

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1 Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses Elissa J. Schwartz Associate Professor School of Biological Sciences Department of Mathematics & Statistics Washington State University

2 Equine Infectious Anemia Virus (EIAV) Establishes a chronic, persistent infection

3 Equine Infectious Anemia Virus (EIAV) Establishes a chronic, persistent infection Recurrent episodes Fever High viral loads Thrombocytopenia

4 Equine Infectious Anemia Virus (EIAV) Establishes a chronic, persistent infection Recurrent episodes Fever High viral loads Thrombocytopenia Similarities to HIV Lentivirus Virus structure, genome, life cycle

5 Equine Infectious Anemia Virus (EIAV) Establishes a chronic, persistent infection Recurrent episodes Fever High viral loads Thrombocytopenia Similarities to HIV Lentivirus Virus structure, genome, life cycle No immunodeficiency

6 Equine Infectious Anemia Virus (EIAV) Establishes a chronic, persistent infection Recurrent episodes Fever High viral loads Thrombocytopenia Similarities to HIV Lentivirus Virus structure, genome, life cycle No immunodeficiency Control: both antibodies and CTLs

Virion and genome EIAV563 Equine Infectious Anemia in 2014 HIV Fig. 1. (A) The equine infectious anemia virion structure showing location and identity of structural proteins.

7 Virion and genome EIAV563 Equine Infectious Anemia in 2014 HIV Fig. 1. (A) The equine infectious anemia virion structure showing location and identity of structural proteins. (B) The 8 kbp equine infectious anemia virus (EIAV) provirus is shown, with long terminal repeats (LTR) and protein-coding regions (gag, pol, env, tat, S2, and rev), with protein names added. (C) Major protein antigens of EIAV used in current commercial test kits (all p26 based; one commercial ELISA kit includes p26 and a determinant of Fig. 1. (A) The equine infectious anemia virion structure showing location andgp45, identitybut of no discrimination is made). The immunoblot test is mainly a research test today, structural proteins. (B) The 8 kbp equine infectious anemia virus (EIAV) provirus is shown, can with long terminal repeats (LTR) and protein-coding regions (gag, pol, env, but tat, S2, anddetect immune responses against all 3 major proteins of EIAV. AGID, agar gel immu-

8 Issel et al. (2014) Vet Clin Equine 30: Stages of infection

9 Stages of infection Escape Issel et al. (2014) Vet Clin Equine 30:

10 Stages of infection Escape Escape Issel et al. (2014) Vet Clin Equine 30:

11 Advantages of studying EIAV Large animal model of persistent viral infection The host mounts an adapting and effective immune response, even with escape Data Clinical studies Experimental infections In vitro experiments

12 Viral dynamics of EIAV infection Goals: (with Naveen Vaidya, Karin Dorman, Susan Carpenter, and Bob Mealey) To estimate the the kinetics of EIAV infection in vivo without immune responses To quantify the effect of neutralizing antibodies in preventing infection Methods: Experimental data on viral load over time in horses infected with EIAV were fitted to the basic mathematical model of viral dynamics Used SCID horses (with naturally occurring severe combined immunodeficiency) to examine viral dynamics in animals with no adaptive immune responses Outcomes: Ø We estimated the rates of Ø infection Ø virus production Ø virus clearance Ø infected cell death Ø Our estimated parameters were used to calculate Ø the basic reproduction number Ø virus doubling time Ø minimal efficacy of antibodies that blocked infection

13 Horse viral load data virus virus Horses were experimentally infected with EIAV Viral load was measured frequently in acute infection SCID = severe combined immunodeficiency Taylor et al. (2010) Journal of Virology 84(13):6536. Mealey et al. (2008) Vet Immunol Immunopathol 121:8-22. No T or B cells à no CTL or antibody responses are made by horses

14 Model dm = λ ρm βmv dt di = βmv (δ + σ )I dt dv = bi (γ + α)v dt M = I = V = Uninfected target cells (macrophages) Infected cells Virus

15 Model dm = λ ρm βmv dt di = βmv (δ + σ )I dt dv = bi (γ + α)v dt l = uninfected cell recruitment rate r = uninfected cell death rate b = infection rate d = infected cell death rate b = virus production rate g = virus clearance rate s =killing of infected cells by CTLs (cytotoxic T lymphocytes) a = virus clearance by antibodies

16 Model fit to data Schwartz EJ, Vaidya NK, Dorman KS, Carpenter S, and Mealey RH. (2018) Virology 513:

17 Parameter Infection rate b ( 10-7 ) ml/(vrna Infected cell death rate d Virus production rate b vrna copies/ Model parameter estimates copies*day) day -1 (cell*day) Virus clearance rate g day -1 With d as a fitted parameter Horse 0.94 ( ) 0.06 ( ) 2124 ( ) ( ) A2245 A ( ) 0.06 ( ) 889 ( ) ( ) A ( ) 0.05 ( ) 870 ( ) ( ) A ( ) 0.06 (0 0.58) 746 ( ) 9.01 ( ) A ( ) 0.06 ( ) 1294 ( ) 9.60 ( ) A ( ) 0.07 ( ) 507 ( ) ( ) A ( ) 0.07 ( ) 101 (91 108) 2.85 ( ) Median (IQR) 3.20 ( ) 0.06 ( ) 870 ( ) ( ) With d = r = 1/21 Horse 0.94 ( ) 1/ ( ) ( ) A2245 A ( ) 1/ ( ) 6.73 ( ) A ( ) 1/ ( ) ( ) A ( ) 1/ ( ) 8.02 ( ) A ( ) 1/ ( ) 2.82 ( ) A ( ) 1/ ( ) 2.33 ( ) A ( ) 1/21 92 (86 97) 2.94 ( ) Median (IQR) 3.25 ( ) 1/ ( ) 6.73 ( )

18 Parameter identifiability Two fixed parameters, r and l based upon estimates derived from previous experimental studies r calculated from estimate of macrophage lifespan of 21 days (Valli 2007) l calculated from r and M 0, where M 0 determined from monocyte count (Mealey 2008) and differentiation rate into macrophages (Hasegawa 2009) s = 0, a = 0 With l fixed and 8 viral load data points, 5 parameters can be uniquely identified (Wu et al. 2008) In this study: Average number of data points per horse = 11 Parameters to identify in our model = 4 à Parameters are identifiable

19 Other kinetic estimates calculated The basic reproductive number (R 0 ) was calculated to be 18.8, which falls within the ranges reported for HIV (6 19) and SIV (4 37). The EIAV doubling time was calculated to be 1.0 day, which is not far greater than that reported for HIV (0.7 day) Steady state levels of EIAV viral load (10 6 ) were within an order of magnitude of HIV

20 Sensitivity analysis Estimated values do not vary greatly compared to previous estimates

21 Virus Escape from Antibody Responses Passive transfer of neutralizing antibodies before and during EIAV infection Horses were infused with EIAVspecific antibodies (NAbs) on days -1, 7, 14 Infection of horses with severe combined immunodeficiency (SCID) No T or B cells à no CTL or antibody responses are made naturally FIG. 3. Peripheral blood platelet counts (open squares) and plasma viral loads (filled circles) for SCID foals that received A2150 convalescent immune plasma (experimental) (a to d) and normal horse plasma (controls) (e to h). Stars indicate febrile days (rectal temperature of F). Platelet counts were not obtained at missing data points. Taylor et al. (2010) Journal of Virology 84(13):6536.

22 Other kinetic estimates calculated, cont. We calculated infectivity to be 2.05 x 10-3 infected cells per RNA copy, similar to 3.8 x 10-4 infected cells per RNA copy in vitro for EIAV (Wu et al. 2011) We also calculated the minimal efficacy of antibodies that blocked infection: We estimated that the effect of infused neutralizing antibodies on clearing the virus and preventing infection in an immunized naïve horse (a ) was 17.8 times higher (median value;; range 9.6 to 22.3) than the virus clearance rate (g ). (Via comparison of R 0 between unprotected horses (R 0 > 1) and the protected horse (R 0 < 1))

23 Implications The leveling off of virus replication in EIAV-infected SCID horses implies that factors other than adaptive immune responses limit the viral growth, such as target cell limitation and/or innate immune responses Results on d r imply that EIAV infection in SCID horses is at most mildly cytopathic (less cytopathic that other lentiviruses studied in immunocompetent hosts) This minimal efficacy of infused antibodies that successfully prevented infection may be useful in the development of therapies and vaccines: When antibody neutralization is on the order of 17.8-fold greater than viral clearance, it can be sufficient to block infection. These results may have implications for the control of other viral infections, such as HIV.

24 Summary of EIAV kinetics in SCID horses This work estimates viral dynamics of EIAV infection in vivo without adaptive immune responses Rates estimated include the infection rate, death rate of infected cells, virus production rate, and virus clearance rate Estimated parameters were used to calculate the basic reproductive number, the virus doubling time, the exponential growth rate, and steady state levels of uninfected cells, infected cells, and virus This work quantifies the functional effect of infused antibodies in preventing infection

25 Acknowledgements Naveen Vaidya, San Diego State University Bob Mealey, Washington State University Susan Carpenter, Iowa State University Karin Dorman, Iowa State University

Equine Infectious Anemia

Equine Infectious Anemia CJ Issel, DVM, PhD University of Kentucky Gluck Equine Research Center Typical Clinical Course of EIAV Infections Acute Chronic Inapparent 42 10 9 10 8 41 200 Viral RNA log 10