Christine S. Rollier

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

ABSTRACT Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Protective and therapeutic effect of DNA-based immunization against hepadnavirus large envelope protein ☆ ☆☆

BACKGROUND & AIMS Studies in the murine model suggest that injection of DNA encoding hepatitis B virus structural proteins is promising for the induction of a specific immune response. We used the duck hepatitis B virus (DHBV) model to study the protective and therapeutic effects of naked DNA immunization against hepadnaviral large envelope protein. METHODS A pCI-preS/S plasmid expressing the DHBV large protein was used for intramuscular immunization of ducks. The humoral response was tested by enzyme-linked immunosorbent assay, immunoblotting, neutralization, and in vivo protection tests. For DNA therapy, DHBV-carrier ducks received four injections of this plasmid. Viremia was monitored for 10 months; thereafter, liver biopsies were performed. RESULTS Immunization with pCI-preS/S plasmid induced a specific, long-lasting, neutralizing, and highly protective anti-preS humoral response in uninfected animals. After pCI-preS/S treatment, a significant and sustained decrease in serum and liver DHBV DNA was observed for carrier ducks compared with the controls. CONCLUSIONS DNA immunization against DHBV large protein results in a potent and protective anti-preS response in the duck model. The results of long-term follow-up of DNA-treated chronically infected ducks are promising and show the usefulness of this model for the study of genetic immunization in chronic hepatitis B therapy.

CD8+ T Effector Memory Cells Protect against Liver-Stage Malaria

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8+ T cells into effector, effector memory (TEM), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and TEM response that requires long intervals for an efficient boost. A preferential TEM phenotype was maintained in liver, blood, and spleen after Ad/MVA prime–boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8+ TEM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific TEM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 TEM populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

Long-Term Thermostabilization of Live Poxviral and Adenoviral Vaccine Vectors at Supraphysiological Temperatures in Carbohydrate Glass

A sucrose-trehalose glass film dried onto a filter can preserve the activity of two potential live viral vaccine vectors at elevated temperatures for up to 6 months. Candy-Coated Vaccines According to the Gates Foundation, improved vaccines are key to progress in global health. As they state on their Web site, “Millions of lives could be saved in the developing world by creating new vaccines that are effective after a single dose, that can be delivered without needles, and that do not require refrigeration.” This work by Alcock et al. addressed the last issue: the thermostability of vaccines. They selected two viral vectors that are promising candidates for developing-world vaccines and stabilized them in a glassy film made of sugars. The viruses retained infectivity and were immunogenic for 6 months at temperatures up to 45°C. As liquids are cooled or water is evaporated from solutions, the component molecules can form a glass, defined as a substance that has been cooled to a solid state without forming crystals. When in this state, sugars—particularly trehalose—can stabilize embedded biological molecules, whether proteins or lipids, likely because of their restricted mobility and tendency to form stabilizing hydrogen bonds with exposed hydroxyl groups. Alcock and colleagues have taken advantage of this well-known property of sugar glasses and applied it to two viruses that are being adapted as vaccines for the developing world—vaccinia virus and adenovirus. They deposited concentrated sugar solutions containing these viruses on filter-like disks and let them dry at room temperature. The viruses recovered from the disks immediately after drying were still able to infect mice and raise immune responses. By keeping the sugar-coated viruses progressively longer and at higher temperatures, the authors tested the ability of the sugar glass to stabilize the viruses in conditions closer to those encountered by a real vaccine preparation. The vaccinia virus was generally quite a bit more stable than the adenovirus, but even the adenovirus retained full infectivity and immunogenicity after 6 months at 45°C on one type of filters. After 1 month of storage at 25°C on this same kind of filter—but without the sugar glass—the adenovirus had lost all potency. Preserving actual, effective vaccines during an extended trip from manufacturing plant to delivery site will no doubt require further optimization. But one other feature of this method may help. The filters coated with sugar can be easily fitted into a holder that attaches to a syringe for shipment and delivery. Now we just have to find a vaccine that is effective after just one dose and that can be administered without a needle. Live recombinant viral vectors based on adenoviruses and poxviruses are among the most promising platforms for development of new vaccines against diseases such as malaria, tuberculosis, and HIV-AIDS. Vaccines based on live viruses must remain infectious to be effective, so therefore need continuous refrigeration to maintain stability and viability, a requirement that can be costly and difficult, especially in developing countries. The sugars sucrose and trehalose are commonly used as stabilizing agents and cryoprotectants for biological products. Here, we have exploited the ability of these sugars to vitrify on desiccation to develop a thermostabilization technique for live viral vaccine vectors. By slowly drying vaccines suspended in solutions of these disaccharide stabilizers onto a filter-like support membrane at ambient temperature, an ultrathin glass is deposited on the fibers of the inert matrix. Immobilization of two recombinant vaccine vectors—E1/E3-deleted human adenovirus type 5 and modified vaccinia virus Ankara—in this glass on the membranes enabled complete recovery of viral titer and immunogenicity after storage at up to 45°C for 6 months and even longer with minimal losses. Furthermore, the membrane carrying the stabilized vaccine can be incorporated into a holder attached to a syringe for almost simultaneous reconstitution and injection at point of use. The technology may potentially be developed for the deployment of viral vector–based biopharmaceuticals in resource-poor settings.

Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection

ABSTRACT Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.

Vaccine-induced early control of hepatitis c virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity

Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV‐specific immune responses, and that all vaccinees exposed to heterologous chimpanzee‐adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P = 0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA‐4 (1) and PD‐1 levels in liver correlated with clearance or chronicity. Conclusion: Despite early control of virus load, a virus‐associated tolerogenic‐like state can develop in certain individuals independent of vaccination history. (HEPATOLOGY 2007;45:602–613.)

Chronic hepatitis C virus infection established and maintained in chimpanzees independent of dendritic cell impairment

Chronic hepatitis C virus (HCV) infection in humans is associated with an impairment of dendritic cells (DC). It has been hypothesized that impairment of DC function may be a central mechanism facilitating the establishment of a chronic carrier state. However, the majority of patients studied with DC impairment to date have been identified and, thus, inadvertently selected because of clinical manifestations leading to their diagnosis, which may have been many years following actual infection. We set out to determine whether impaired DC function occurred in the earlier asymptomatic phase of infection and turned to a well‐defined cohort of HCV‐infected chimpanzees in which the specific date of infection and the nature of the inoculum were well characterized. Results revealed that, in contrast to the observations in human subjects with advanced clinical hepatitis, there was neither impairment of the allostimulatory capacity of monocyte‐derived DC from HCV chronic carriers nor impairment of the maturation process. Decreased allostimulatory capacity was only detected in 2 animals and, interestingly, in those that possessed the highest viral loads. Nevertheless, HCV sequences were undetectable in any of the DC derived from HCV‐infected chimpanzees. In conclusion, these findings suggest that the mechanisms of establishing persistent HCV infection are separate and independent from those responsible for impaired DC function. Indeed, the maturation and allostimulatory impairment, as described in patient studies, are not necessary prerequisites but rather possible consequences of persistent and active HCV infection associated with disease progression. (Hepatology 2003;38:851–858).

Immune responses against a liver-stage malaria antigen induced by simian adenoviral vector AdCh63 and MVA prime-boost immunisation in non-human primates.

Malaria is a major health problem as nearly half of the human population is exposed to this parasite causing around 600 million clinical cases annually. Prime-boost regimes using simian adenoviral vectors and MVA expressing the clinically relevant Plasmodium falciparum ME.TRAP antigen have shown outstanding protective efficacy in mouse models. We now extend those observations to macaque monkeys. Immunisation with AdCh63 elicited a median response of 869 IFN-γ SFC/million PBMCs to ME.TRAP and responses were boosted by MVA to reach 5256 SFC/million PBMCs, increasing at the same time the breadth of the T cell responses to cover the complete ME.TRAP antigen. Intramuscular vaccination was more immunogenic than the intradermal route, and MVA could be used repeatedly for up to 3 times to boost adenovirus-primed responses. An interval of 16 weeks between repeated MVA injections was optimal to enhance cytokine production by T cells and improve the CD8 multifunctional responses. Antibodies to TRAP were exceptionally high and maintained for a long period of time after the prime-boost regime. These results in non-human primates highlight the potential of this vaccination regime and encourage its future use in clinical trials.

Progress in DNA vaccine for prophylaxis and therapy of hepatitis B.

Increasing lines of evidence suggest that DNA vaccine is of interest to fight chronic hepatitis B virus (HBV) infection. We used the Pekin duck infected by duck HBV (DHBV), closely related to the human virus, which is an attractive model allowing study of protective and therapeutic effectiveness of DNA vaccines against hepatitis B. Immunisation with a plasmid encoding the DHBV large (L) envelope protein induced a strong, specific, highly neutralising and long-lasting anti-preS humoral response in uninfected ducks. Importantly, maternal antibodies elicited by such DNA immunisation were vertically transmitted and protected progeny against viral challenge. Therapeutic immunisation of chronic DHBV-carrier ducks with this plasmid DNA led to the dramatic and sustained decrease in viral replication and even to clearance of intrahepatic viral covalently close circular DNA (cccDNA) pool in some animals. Our recent combination therapy data showed even a more pronounced antiviral effect of DNA vaccine to DHBV envelope protein when associated with antiviral drug (lamivudine) treatment. Therefore, DNA-based vaccine appears as a promising new approach for prophylaxis and therapy of hepatitis B.

Maternally Transferred Antibodies from DNA-Immunized Avians Protect Offspring against Hepadnavirus Infection

The outcome and protective efficacy of maternal antibodies elicited by DNA immunization to the large (L) hepadnavirus envelope protein were studied using the duck hepatitis B virus (DHBV) model. Following genetic immunization of breeding ducks with a DHBV L protein gene-bearing plasmid, specific and highly neutralizing antibodies were transferred from the sera of immunized ducks, via the egg yolk, to the progeny of vaccinees. Interestingly, large amounts (60 to 100 mg/egg) of high-titer and L protein-specific yolk immunoglobulins (immunoglobulin Y) accumulated in the egg yolk. These results suggest that eggs from genetically immunized avians may represent a potent source of DNA-designed antibodies specific to viral antigen. Importantly, these antibodies are vertically transmitted and protect offspring against high-titer DHBV challenge.