T-cell Immunity to the Hepatitis C Virus During and After Pregnancy BCMM AND VACCINES & IMMUNITY JOINT MEETING Sept 2, 2011 When thinking about the problem of vertical tranmission and the lack of preventative interventions, there are many biological questions that arise. How and when does transmission occur – via blood mixing in the placenta, or through mucosal surfaces as the baby passes through the bloody vaginal canal? Is it free virus, or cell-associated virus that is more important? Why is it that 95% of the infants do not get infected? I started on this project with Mona to study vertical transmission several years ago as a fellow in Dr. Chris Walker’s laboratory. Dr. Walker’s lab has been a big part of showing the importance of T-cell immunity in the control of HCV. This led us to approach the topic with regard to T-cell immunity. Thus I will talk about two aspects of T-cell immunity in this population – that of the infants, and that of the mothers.

Hepatitis C Virus Small, positive-stranded RNA virus Prototype virus within the Hepacivirus genus of the Flaviviridae C E1 E2 p7 NS2 NS3 aNS4b aNS5b ≈3000 aa Envelope Glycoproteins Core Serine protease Helicase Protease Cofactor RNA dependent RNA polymerase F First we’ll step back to review basics of HCV. For those of you less familiar with the virus, hepatitis C is a positive stranded RNA virus in the flaviviridae family. Its genome codes for a single polyprotein that is cleaved into 10 component proteins by host and viral proteases. A recent count estimated the global prevalence to be approximately 2.2%. Transmission occurs principally by contact with contaminated blood. Unscreened blood transfusions and re-use of improperly sterilized needles have been major factors worldwide. Sharing needles during IVDU is the most common route in the United States. --------------- P7 - ?ion channel, ?fxn in particle assembly and release or viral entry NS2 - cis acting cleavage from NS3. -- fxn uncertain NS4b - helps assemble replication complex?? GTPase?? NS5a - hypo or hyperphosphorylated. Cell cx adaptions in NS5a render hypophosphoyrlated. -- interacts with other NS proteins in replication complex, also with host proteins in replication complex (Snare-VAP-A, FBL2) and with viral RNA -- interacts with cellular proteins such as PKR (Interferonsensitivity determining region)--may play a role in interferon sensitivity -- interacts with tumor suppression proteins -- large section may be deleted and virus still replicates in culture. 6 Genotypes, multiple subtypes Genotype 1 is the most common and the most resistant to treatment

Outcome of HCV Infection Resolved Infection (25%) Normal Liver Acute Infection Chronic Infection (75%) Chronic Hepatitis Cirrhosis (20%) Carcinoma (1-4% per year) Rapid Progression Alcohol use, coinfection ≤ 20 years Upon infection, the virus replicates primarily in the liver. Up to 25% of acutely infected individuals are able to clear the virus, but the majority develop lifelong infection with 20% developing cirrhosis over 20-30 years and a portion of those developing hepatocellular carcinoma. HCV related liver diseases are the #1 indication for liver transplantation in the developed world. Lauer & Walker NEJM 2001 The course of infection varies widely among persons. Factors that decrease the risk of progression include female sex and a younger age at infection; factors that increase the risk include alcohol intake, an older age at infection, male sex, and coinfection with other viruses. Persons with a favorable risk profile often do not have progressive liver disease until 30 or more years after infection. In contrast, 20 percent of persons with chronic hepatitis C will eventually have cirrhosis, and this can occur 20 years or less after infection, especially in those with alcohol abuse or coinfection with human immunodeficiency virus type 1 or hepatitis B virus. Once cirrhosis is established, the risk of hepatocellular carcinoma is 1 to 4 percent per year. ≥ 30 years Slow Progression Female sex, young age at infection - HCV related liver diseases – now the leading cause for liver transplantation in developed world Adapted from Lauer G & Walker B. NEJM 2001;345:41-52

T-cell Immunity to HCV in Acute Resolving vs Persisting Infection ~25% ~75% Transaminase (liver cell death) 2 6 12 24 Viremia CD4+ T cells (blood) CD8+ T cells (blood) serum antibodies weeks years Rapid Resolution Persistent Infection T-cell immunity is known to be an important factor in determining the outcome of infection in adults and animal models. These figures illustrate a typical course of acute resolving and persisting HCV infections in humans and chimpanzees (the only animal model of persistent infection). Top panel. Viremia (indicated by the brown line) usually peaks at 8-12 weeks after infection in acute resolving hepatitis C. Expansion of virus-specific CD4+ and CD8+ T cells in blood and their infiltration into liver is temporally associated with a decline in viremia and rise in serum transaminases. Successful T cell responses fully contract only after apparent resolution of infection. Bottom panel. HCV infections that persist are characterized by variable T cell responses that often target fewer epitopes and reach lower peak frequencies. HCV-specific CD4+ T cell responses either never develop or fail before virus is cleared. CD8+ T cells are sometimes detected in blood for weeks to months following infection but can persist in the chronically infected liver for decades. Durable serum antibody responses to viral proteins develop early in infection. Neutralizing antibodies also appear but at least initially have a limited capacity for recognition of rapidly emerging HCV variants.

(Nikolich-Zugich et al. Nat Rev Imm. 2004; 4:123-132) T-Lymphocytes: Adaptive Immune Cells Randomly generated receptors - Repertoire of 2*107 distinct T-cell receptors in peripheral blood Recognize foreign peptides presented by MHC molecules on cell surfaces CD8+ Cytotoxic T-cells: Target peptides from endogenous proteins on infected cells Kill infected cells CD4+ Helper T-cells: Target peptides from exogenous proteins presented by professional antigen presenting cells Secrete antiviral cytokines and augment function of CD8+ T-cells and B-cells (Nikolich-Zugich et al. Nat Rev Imm. 2004; 4:123-132)

HCV-specific CD8+ T-cell HCV-Infected Hepatocyte Class I MHC Molecule HCV Peptide T-cell Receptor Antiviral cytokines inhibit viral replication & cytotoxic chemicals kill infected cells T-cell Success in Resolving HCV T-cells recognize short peptides from foreign proteins that are displayed on the surface of cells. In the case of HCV, this lower cell would be an HCV infected hepatocyte. The infected cell has mechanisms to digest HCV proteins into short peptides. These peptides are complexed to MHC molecules and displayed on the cell surface. If a roaming T-cell has a receptor specific for that peptide in the HLA molecule, it will bind, triggering signals, which in the case of Cytotoxic, or CD8+ T-cells results in the release of granules capable of directly killing the infected cell, as well as release cytokines such as interferon-gamma which turns on antiviral genes in surrounding cells.

T-cell Failure in Chronic HCV Escape mutation T-cell exhaustion Mutated HCV Epitope Poor Proliferation Reduced cytotoxicity and antiviral cytokine secretion Low Frequency Inhibitory co-receptors (PD-1, CTLA4, Tim-3) HCV-specific CD8+ T-cell HCV-specific CD8+ T-cell One way that HCV subverts the adaptive immune response is by accumulating amino acid substitutions in CD8+ epitopes that prevent binding to the MHC molecule or recognition by the TCR. In cases where the T-cell epitopes remain intact, the HCV-specific T-cells are often dysfunctional. As I said before, they tend to be of low frequency. In in-vitro testing they proliferate poorly and have impaired cytotoxicity and antiviral cytokine secretion. It has recently been found that these T-cells express inhibitory co-receptors that negatively regulate their response to antigens. Sorting this failure T-cells in chronic HCV is an area of active investigation. Many efforts are underway to see if T-cell exhaustion can be reversed. HCV-Infected Hepatocyte HCV-Infected Hepatocyte

Weak HCV-specific T-cell responses in chronic infection HCV 1b Peptide Set Core E1 E2 P7 NS2 NS3 aNS4b aNS5b CMV pp65 DMSO control to illustrate an example of the dysfunctional T-cell response to HCV, this shows the IFN-g ELISpot response of a 24 yo female infected with HCV for over a year. In this assay, 200,000 blood mononuclear cells are incubated with pools of synthetic peptide corresponding to the proteins of HCV. Each spot is a footprint of a T-cell that secreted interferon gamma in response to the HCV peptides. In this case no significant T-cell responses to HCV were detected. The T-cell defect in HCV is specific to HCV. As you can see she had a strong response to peptides from a CMV protein. Freshly isolated peripheral blood mononuclear cells were incubated for 36 hours in the ELISpot plates along with HCV peptides. The peptides were divided into 9 pools spanning the entire HCV polyprotein as shown here. At top are the controls, PHA as a positive control, and GFP and no-peptide as negative controls. Each condition is performed in duplicate. Each spot represents an individual interferon-secreting cell. This particular plate is from a blood draw at 32 weeks gestation and shows a subtle but significant T-cell responses against NS3 and NS5 proteins. Ex-vivo IFN-γ ELISpot 200,000 PBMCs/well

OSU-NCH Hepatitis C Virus Immunity in Women and Children Study Antepartum Maternal Samples infant Samples 18 mo Delivery 6 mo 3 mo Our interest in immunity to HCV during pregnancy has been triggered by several observations made during an ongoing study that is looking at vertical transmission of HCV. Vertical transmission occurs in 5% of pregnancies to viremic women. Several questions being asked in this study are whether maternal or infant T-cell responses are protective, whether the 95% of “uninfected” children had detectable HCV-specific T-cell responses, and whether viral persistence in the infected children is associated with viral mutations that allow it to escape from T-cell immunity. We’ve set up a collaboration between …. We are enrolling HCV-infected pregnant women and collecting blood samples from them during pregnancy and from them and the infant at delivery, 3 mo, 6 mo, and 18 months post-partum for assays that would look at the virus and T-cell immunity to the virus. In the course of this project, we noted several interesting trends in maternal immunity during and after pregnancy, which will be the focus of the rest of the discussion.

Influence of Pregnancy on Hepatitis C Viral Load M001: Genotype 2b. Age at 1st delivery: 26 yrs. Estimated duration of infection prior to 1st delivery: 12 yrs. M003: Genotype 1a. Age at 1st delivery: 34 yrs. Estimated duration of infection prior to 1st delivery: 0.6 yrs. M016: Genotype 2a. Age at 1st delivery: 24 yrs. Estimated duration of infection prior to 1st delivery: 4.7 yrs.

Influence of Pregnancy on Hepatitis C Viremia Years Viremia CD8+ Function CD4+ Function Prepregnancy Pregnancy Postpartum Hypothesis: Resurgent HCV-specific T-cell immunity after delivery mediates the drop in viremia.

Postpartum Viral Load Decline Associated with Broadening of HCV-Specific T-cell Response after Delivery

Resurgence of Polyfunctional CD4+ T-cells Subject M001

Define the Immunological Signature of Postpartum Viral Control Pregnancy Postpartum Postpartum Viral Control Stable Viremia Function & Phenotype of HCV-specific T-cells (Proliferation, Cytokine Secretion, Survival and Inhibitory Receptor Expression) HCV-specific T-cell Receptor Analysis (Diversity, Avidity) Serum Cytokine Profile Gene-expression profile of HCV-specific T-cells

Influence of Pregnancy on HCV Evolution HCV genome mutates readily 1012 virions produced daily RNA-dependent RNA polymerase lacks proofreading function HCV mutates to escape CD8+ T-cell pressure 50-70% of targeted class I epitopes mutate to escape T-cell responses Appearance of escape mutations linked to failure to clear viremia Some escape mutations impair replicative fitness and revert to wild-type when transferred to other individuals Hypothesis: Enhanced CD8+ T-cell pressure after delivery will cause accelerated viral evolution

Viral Sequencing Through Consecutive Pregnancies Subject M003

Viral Sequencing Through Consecutive Pregnancies Study week Preg #1 -6 25 40 56 66 86 152 Preg #2 Vertical lines represent amino acid substitutions relative to week -6 consensus sequence. Height of lines proportionate to fraction of clones bearing mutant residue.

Viral Evolution Accelerates After Both Pregnancies

Reversion of Mutation During 2nd Pregnancy Study week Preg #1 -6 25 40 56 66 86 152 Preg #2

(*Adjusted for transfection efficiency) Mutations in overlapping T-cell epitopes permit escape from T-cell pressure M003 1395/9 M003 1402/9 (*Adjusted for transfection efficiency)

Infectious Virus Production “wk -6 wild-type sequence” Infectious HCV cell culture virus H77S.3 bearing “wild-type” week -6 sequence is more replicatively fit than virus bearing the escape mutant sequences. RNA Replication Infectious Virus Production “wk -6 wild-type sequence” I have a first set of viral fitness data which I presented at our lab meeting last Monday. Attached are the slides for that experiment. The first one is from Rob’s result file for introduction and the second slide is my data. This was obtained from a single experiment and I will get a second set by this weekend. In addition, I am planning to do transfection for the third set of experiment this Friday.

Influence of Pregnancy on HCV Evolution Years Viremia CD8+ Selection Pressure Viral Replicative Fitness Prepregnancy Pregnancy Postpartum A B C A: Reversion of escape mutations B: Re-emergence of previous +/- appearance of new escape mutations C: Compensatory mutations Aim 2. Define patterns of HCV evolution during and after pregnancy. Determine if reduced virus replication after pregnancy is associated with an increased frequency of escape mutations in class I epitopes. Determine the fitness cost of non-synonymous viral escape mutations that are lost during pregnancy. Rationale. Provides an independent virologic readout of intrahepatic CD8+ T-cell selection pressure to address the important question of whether HCV-specific T-cells can be functionally restored Provide insight into the replicative fitness of viral quasispecies passed vertically in mother to child transmission, the most common route of pediatric HCV infection

Pregnancy and other Persistent Viral Infections HBV HIV Mean HBV DNA levels rise in pregnancy and fall in the postpartum period ter Borg et al. J Viral Hepat 2008; 15:37-41 5 of 31 HBeAg+ women became HBeAg- in the postpartum period compared to 0 of 30 non-pregnant women. Lin et al. J Med Virol 1989; 29:1-6 No significant change in viral load during or after pregnancy Burns et al. Am J Obstet Gynecol 1998; 178:355-9 Melvin et al. J Acquir Immune Defic Syndr 1997; 14:232-236 Garcia et al. N Engl J Med 1999;341:394-402 T1 or T2 T3 2 mo PP 12 mo PP 24 mo PP ter Borg et al. Burns et al.