J Infect Dis. 2020 Jan 29. pii: jiaa036. doi: 10.1093/infdis/jiaa036. [Epub ahead of print]
Blocking entry of hepatitis B and D viruses to hepatocytes as a novel immunotherapy for treating chronic infections.
Maravelia P1, Frelin L1, Ni Y2, Pérez NC1, Ahlén G1, Jagya N1, Verch G2, Verhoye L3, Pater L1, Johansson M4, Pasetto A1, Meuleman P3, Urban S2, Sällberg M1.
Author information
Abstract
BACKGROUND:
Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T cell response. We therefore designed an immunotherapy driven by naïve healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry.
METHODS:
Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV- specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes.
RESULTS:
The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice.
CONCLUSION:
We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation.
DiscussionThe current antiviral treatments for chronic HBV and HDV infections using NAs, or other smaller molecules,caneffectively suppress the viral loadduring ongoing therapy and slow downdisease progression, but do not fully preventdevelopment ofliver disease[4,9].None of these compounds effectivelyinducein a functional cure, especially when targeting the immune-tolerant chronic carrier[12,37].A functional cure is defined as the stable loss of HBsAg in serum. Newtherapeutic strategiesare needed the overcome, or circumvent, the immune evasion strategies exerted by the virus tostimulatethe host tocontrol the infectionand induce a functional cure [38].We therefore designed an immunotherapy targeting an additional step of the viral life cycle to improve off-therapy responses.The therapy is designed considering two main issues. First, to block HBV and HDV infectionby stimulating endogenousPreS1 receptor blocking antibody production. Second, toavoid using large HBV sequences, as T cellsto HBVantigensare highly dysfunctional in chronic HBV. In order to recruitehealthyT cellsthat do not cross-react with dysfunctional HBV-specific cellsin patients with chronic HBV, we used HDAg as a heterologous T cell carrier. Since >90% of HBV carriers are not coinfected by HDV [5], the [size=13.3333px]Downloaded from [size=13.3333px]https://academic.oup.com/jid/adv ... dis/jiaa036/5717223 by guest on 02 February 2020[size=100px]Accepted Manuscript13HDAg-specific T cells are,in a vast majority ofHBV carriers,as naïve and healthy as those to any other irrelevant heterologous carrier [23]. However, these HDV-specific T cells can still be relevant for HDV and therebyan HBVmono-infected carrier may through the PreS1-HDAg-based vaccine become immune to HDV superinfection. Thus, using HDAg as a carrier for PreS1 sequences serves multiple purposes.Usinga genetic vaccine that containsPreS1 and HDAg,we caninduce anendogenous production ofPreS1 antibodies that block the PreS1-mediated entry of HBVusingthe receptor NTCP [39].PreS1is only a minorcomponenton sub-viral particlesthat cause the immune-dysfunction,but vital for the infectivity of HBVparticles[18,20,40]. Therefore, the PreS1-specific antibodiesefficiently avoid being blocked by the circulating sub-viral particles and canneutralize HBV [19].Totest our concept described above, we generated tendifferentDNA plasmids as immunogens containing various combinations of PreS1 and/or HDAg sequences. These were tested for induction of specific antibodies and T cellsto identifythe most immunogenicdesign. TheDNA constructs induced strongPreS1and HDV T cell responses. The HDAg-specific T cells weregenotype specificin mice oftwo different genetic backgrounds(Figure 2and data not shown).Thus, to avoid a potential T cell non-responder status in humans,we selectedconstructscarryingtwo strainsof HDAgcorresponding to the two major genotypesof HDV. TheconstructscombiningPreS1 and HDAg sequences effectively induced high levels of PreS1 antibodiesin both mice and rabbits. PreS1antibodies frombothspecieswere highly cross-reactive againstdifferent HBV types and subtypes. Themost immunogenic design,that raised potentHBV/HDV-specific T cell responses in wild-type and HLA-A2 transgenic mice and high anti-PreS1 titers,comprisedHDAg sequences of twogenotypes coupled to tandem PreS1 sequences. Importantly, we could demonstratethat the antibodies induced by these active immunogens were also themost effective at neutralizing HBV in vitro,using a well-established [size=13.3333px]Downloaded from https://academic.oup.com/jid/adv ... dis/jiaa036/5717223 by guest on 02 February 2020[size=100px]Accepted Manuscript14assaythat efficiently supports the full viral life cycle [22,33].Weweretheninterested indeterminingwhether theseentry blocking anti-PreS1 antibodies could inhibitHBV infection ofhuman hepatocytes in vivo. From the studies in rabbits,it was evident that antibody levels>103toPreS1 as determined by ELISA neutralized HBV in vitro.Wetherefore purified total IgG from D4 immunized and non-immunizedrabbitsand evaluated theseantibodies for protection against HBV infection in a mouse modelrepopulated with human hepatocytes[35]. We foundthat a single injection of these antibodies indeedprotected, or limited,HBV infection in vivo over a threetoeight-week period. Thereduced levels in two mice were most likely due to a limited first round infection and neutralization over the first weeks from challenge. Thus, this delayed thedevelopment of peak HBV viremia. We thereforeconcluded that a D4DNA-based immunotherapy protecthuman hepatocytes against HBV infectionboth in vitro and in vivo.There are currently different approaches indevelopment aiming atinhibitingHBV entryand these are mainly based either on PreS1-derivedpeptides or antibodies targeting the S-or PreS1/2domainsof HBsAg. PreS1-antibodies are known toneutralizeHBV[41].The mostadvanced approach in clinical developmentso faris aPreS1peptide-based competitiveinhibitor termed Myrcludex B whichhas been successful in blocking entry of mainly HDV, but also HBV[42]. This peptide, however,requiresdaily subcutaneouslyadministration for a period over12-24 weeks to reach optimalantiviral effects in patients with chronic HDV, and/or HBV. Also, although it can successfullyprevent HDV/HBV entryby competitivelybind to NTCP,alterationsin the bile acid transport and metabolite functions of the receptorhave been noted[43,44]. PreS1-peptide based vaccines have also been shown to be able to break immunotolerance in chronic HBV carrier mice, supporting its important therapeutic role in controlling HBV infection[45]. Other strategies in the pipeline are therapeutic vaccines containing different versionsof HBV antigens[46]. Although, it hasbeen shown that vaccines containing only HBV sequences can be safe and occasionally elicit T cell responsesin chronic [size=13.3333px]Downloaded from https://academic.oup.com/jid/adv ... dis/jiaa036/5717223 by guest on 02 February 2020[size=100px]Accepted Manuscript15HBV carriers [24,25], it still remains unclear ifthey can overcome, or restore,the T cell dysfunctionof the infected hostand,thus,be of clinical benefit[47]. It is likely thatadditional immunologic-triggers are needed to activatethe host ́s immune responses[48,49].Wetherefore aimedat raising endogenously produced antibodies against the PreS1 antigen using PreS1 sequenceslinked to HDAg. HereHDAg actsas a heterologous T cell epitope carrier thatcircumvents, or bypasses,the engagement and dependence on the dysfunctional HBV-specific T cell response present in a chronically infected host. As an added benefit, the immunizedchronic carriers mono-infected by HBV can become immune against an HDV super-infection. In conclusion, we have designed a newimmunotherapyapproachfor HBVand potentially HDV. Ourdesign concept aims atcircumventingthe major hurdles in the infected host, such as overexpression of antigens that block neutralizing antibodies and theprofound T cell dysfunction/tolerance. Here HDAg serves as a healthy heterologous T-and B-cell carrier to circumvent the engagement of dysfunctional HBV-specific T cellsin the induction of PreS1 antibodies. Weshowthat we induceT cells specific for PreS1 and HDAg and antibodies that block HBV entry into hepatocytes in vitro and in vivo. Theconcept of bypassing T cell tolerance willbe further assessed in the context of a chronic carrier model.In conclucion, using thisapproach, with the host as the producer of the entry inhibitors,this concept may in combination with existing and coming maturation inhibitors achieve durable off-therapy responses and a functional cure for chronic HBV infection.