Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles.

UNLABELLED: At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. IMPORTANCE: From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Comparison of the immunogenicity of the human papillomavirus (HPV)-16/18 vaccine and the HPV-6/11/16/18 vaccine for oncogenic non-vaccine types HPV-31 and HPV-45 in healthy women aged 18-45 years.

Protection against oncogenic non-vaccine types (cross-protection) offered by human papillomavirus (HPV) vaccines may provide a significant medical benefit. Available clinical efficacy data suggest the two licensed vaccines (HPV-16/18 vaccine, GlaxoSmithKline Biologicals (GSK), and HPV-6/11/16/18 vaccine, Merck & Co., Inc.) differ in terms of protection against oncogenic non-vaccine HPV types -31/45. The immune responses induced by the two vaccines against these two non-vaccine HPV types (cross-reactivity) was compared in an observer-blind study up to Month 24 (18 mo post-vaccination), in women HPV DNA-negative and seronegative prior to vaccination for the HPV type analyzed (HPV-010 [NCT00423046]). Geometric mean antibody titers (GMTs) measured by pseudovirion-based neutralization assay (PBNA) and enzyme-linked immunosorbent assay (ELISA) were similar between vaccines for HPV-31/45. Seropositivity rates for HPV-31 were also similar between vaccines; however, there was a trend for higher seropositivity with the HPV-16/18 vaccine (13.0-16.7%) versus the HPV-6/11/16/18 vaccine (0.0-5.0%) for HPV-45 with PBNA, but not ELISA. HPV-31/45 cross-reactive memory B-cell responses were comparable between vaccines. Circulating antigen-specific CD4+ T-cell frequencies were higher for the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (HPV-31 [geometric mean ratio [GMR] =2.0; p=0.0002] and HPV-45 [GMR=2.6; p=0.0092]), as were the proportion of T-cell responders (HPV-31, p=0.0009; HPV-45, p=0.0793). In conclusion, immune response to oncogenic non-vaccine HPV types -31/45 was generally similar for both vaccines with the exception of T-cell response which was higher with the HPV-16/18 vaccine. Considering the differences in cross-protective efficacy between the two vaccines, the results might provide insights into the underlying mechanism(s) of protection.

Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine.

To monitor immune status during clinical trials and after vaccine registration, several assays have been developed to measure type-specific human papillomavirus (HPV) serum antibody levels. These include neutralization assays, single epitope-based inhibition immunoassays, and direct enzyme-linked immunosorbent assays (ELISAs). Neutralization assays based on multiple epitopes and independent of vaccine material are considered the 'gold standard' for unbiased assessment of the protective potential of vaccine-induced antibodies. However, their use in large clinical trials is challenging. Here, we compare both the direct ELISA and the single epitope-based inhibition ELISA with the pseudovirion-based neutralization assay (PBNA) for HPV-16/18 antibody responses in vaccinated women enrolled in trials of Cervarix, GSK's cervical cancer vaccine. The direct ELISA, which is based on multiple epitopes, was shown to have a higher degree of sensitivity and correlation with the PBNA when compared with the single epitope-based inhibition ELISA. Among double-positive results, high correlations were observed between the PBNA and the direct ELISA (0.70-0.88 for HPV-16 and 0.82-0.94 for HPV-18) and also with the single epitope-based inhibition ELISA (0.60-0.89 for HPV-16 and 0.57-0.96 for HPV-18) in women aged 15-25 years. The correlation persisted up to 6.4 years after primary vaccination. Similar levels of correlation were observed for adolescents aged 10-14 years and women aged 46-55 years. Therefore, the direct ELISA appears to be an excellent surrogate for neutralizing activity and can be used to evaluate antibody response induced by L1 virus-like particle-based cervical cancer vaccines, regardless of time elapsed after vaccination (up to 6.4 years) and the age of the vaccine recipient.

AS03-adjuvanted H7N1 detergent-split virion vaccine is highly immunogenic in unprimed mice and induces cross-reactive antibodies to emerged H7N9 and additional H7 subtypes.

Avian H7 is one of several influenza A virus subtypes that have the potential to cause pandemics. Herein we describe preclinical results following administration of an investigational H7N1 inactivated detergent-split virion vaccine adjuvanted with the AS03 Adjuvant System. The adjuvanted H7N1 vaccine was highly immunogenic compared to the non-adjuvanted H7N1 vaccine in unprimed mice with less than 100ng of hemagglutinin antigen per dose. In addition, compared to the non-adjuvanted vaccine, the AS03-adjuvanted H7N1 vaccine also induced robust HI and VN antibody responses that cross-reacted with other H7 subtypes, including recently emerged H7N9 virus. These H7 data from the preclinical mouse model add to the existing H5 data to suggest that AS03 adjuvant technology may be generally effective for formulating antigen-sparing detergent-split virion vaccines against intrinsically sub-immunogenic avian influenza A virus subtypes.

Antibody avidity measurements in recipients of Cervarix vaccine following a two-dose schedule or a three-dose schedule.

The HPV-16/18 vaccine (Cervarix) is a prophylactic vaccine for the prevention of cervical cancer and contains recombinant virus-like particles (VLPs) assembled from the L1 major capsid proteins of human papillomavirus (HPV) strains 16 and 18. Although a correlate of protection has yet to be identified, HPV-specific antibodies are thought to prevent virus infection of the genital mucosa. Therefore, antigen-specific antibodies as assessed by ELISA or pseudovirion-based neutralisation assay are frequently measured in clinical trials to substantiate the immune responses induced by the vaccine. Measuring antigen-antibody binding avidities, which reflects the degree of affinity maturation in the B-cells, is another valuable method to assess the quality of the antibody responses. Here we describe the antigen-specific antibody avidities in samples taken from a clinical trial examining the feasibility of adopting a two-dose (Months 0 and 6) schedule for 9-14 year olds instead of the three-dose schedule (Months 0, 1 and 6). Antibody avidity (i.e. avidity index [AI]) was determined in the ELISA by the ratio of antibody concentrations in serum samples treated or not with the chaotropic agent NaSCN. Importantly, in the comparison between the groups of two-dose and three-dose recipients, no differences in AIs were observed at Months 7, 24 and 48. The results suggest that from Month 7 to 48, the quality of the antibody response in terms of avidity was similar in the two-dose recipients to that in the three-dose recipients. Hence these results support the adoption of a two-dose schedule in 9-14 year-old girls.

Effects of varying antigens and adjuvant systems on the immunogenicity and safety of investigational tetravalent human oncogenic papillomavirus vaccines: results from two randomized trials.

BACKGROUND: A prophylactic human papillomavirus (HPV) vaccine targeting oncogenic HPV types in addition to HPV-16 and -18 may broaden protection against cervical cancer. Two Phase I/II, randomized, controlled studies were conducted to compare the immunogenicity and safety of investigational tetravalent HPV L1 virus-like particle (VLP) vaccines, containing VLPs from two additional oncogenic genotypes, with the licensed HPV-16/18 AS04-adjuvanted vaccine (control) in healthy 18-25 year-old women. METHODS: In one trial (NCT00231413), subjects received control or one of 6 tetravalent HPV-16/18/31/45 AS04 vaccine formulations at months (M) 0,1,6. In a second trial (NCT00478621), subjects received control or one of 5 tetravalent HPV-16/18/33/58 vaccines formulated with different adjuvant systems (AS04, AS01 or AS02), administered on different schedules (M0,1,6 or M0,3 or M0,6). RESULTS: One month after the third injection (Month 7), there was a consistent trend for lower anti-HPV-16 and -18 geometric mean antibody titers (GMTs) for tetravalent AS04-adjuvanted vaccines compared with control. GMTs were statistically significantly lower for an HPV-16/18/31/45 AS04 vaccine containing 20/20/10/10 µg VLPs for both anti-HPV-16 and anti-HPV-18 antibodies, and for an HPV-16/18/33/58 AS04 vaccine containing 20/20/20/20 µg VLPs for anti-HPV-16 antibodies. There was also a trend for lower HPV-16 and -18-specific memory B-cell responses for tetravalent AS04 vaccines versus control. No such trends were observed for CD4(+) T-cell responses. Immune interference could not always be overcome by increasing the dose of HPV-16/18 L1 VLPs or by using a different adjuvant system. All formulations had acceptable reactogenicity and safety profiles. Reactogenicity in the 7-day post-vaccination period tended to increase with the introduction of additional VLPs, especially for formulations containing AS01. CONCLUSIONS: HPV-16 and -18 antibody responses were lower when additional HPV L1 VLPs were added to the HPV-16/18 AS04-adjuvanted vaccine. Immune interference is a complex phenomenon that cannot always be overcome by changing the antigen dose or adjuvant system.

Cell-mediated immune responses to a varicella-zoster virus glycoprotein E vaccine using both a TLR agonist and QS21 in mice.

Lack of adequate cell-mediated immunity (CMI) to varicella-zoster virus (VZV) has been associated with higher risks of developing herpes zoster (HZ) and associated post-herpetic neuralgia (PHN), and is of particular concern for older and immunocompromised individuals. Thus, the development of an effective HZ vaccine with a clinically acceptable safety profile that is capable of addressing decreased immunity would be highly desirable. In this study we compared the immunogenicity of different vaccine formulations containing VZV glycoprotein E (gE), an important target for CMI and antibody responses, in a VZV-primed mouse model. The formulations included recombinant gE, either unadjuvanted, or combined with aluminium salt or an Adjuvant System (AS01 or AS02), and CMI was used as the primary immunological endpoint. All adjuvanted vaccines induced gE- and/or VZV-specific CD4(+) T cell and antibody responses. A formulation of gE with an Adjuvant System containing the immunostimulants QS21 and 3-O-desacyl-4'-monophosphoryl lipid A (MPL) was shown to be more immunogenic than gE with aluminium salt or unadjuvanted gE (gE/saline). Both immunostimulants were shown to act synergistically in enhancing CMI responses. Formulations with AS01 elicited high frequencies of CD4(+) T cells producing IFN-γ and IL-2. These responses were dose-dependent with respect to both antigen and adjuvant. The gE/AS01(B) candidate vaccine induced higher frequencies of CD4(+) T cells producing IL-2 and/or IFN-γ than all other gE/AS01 formulations, supporting its use for clinical evaluations.

Efficacy of an AS03A-adjuvanted split H5N1 influenza vaccine against an antigenically distinct low pathogenic H5N1 virus in pigs.

We used the pig model of influenza to examine the efficacy of an AS03(A)-adjuvanted split H5N1 (A/Indonesia/05/2005) vaccine against challenge with a low pathogenic (LP) H5N1 avian influenza (AI) virus (duck/Minnesota/1525/1981) with only 85% amino acid homology in its HA1. Influenza seronegative pigs were vaccinated twice intramuscularly with adjuvanted vaccine at 3 antigen doses, unadjuvanted vaccine or placebo. All pigs were challenged 4 weeks after the second vaccination and euthanized 2 days later. After 2 vaccinations, all pigs in the adjuvanted vaccine groups had high hemagglutination inhibiting (HI) antibody titers to the vaccine strain (160-640), and lower antibody titers to the A/Vietnam/1194/04 H5N1 strain and to 2 LP H5 viruses with 90-91% amino acid homology to the vaccine strain (20-160). Eight out of 12 pigs had HI titers (10-20) to the challenge virus immediately before challenge. Neuraminidase inhibiting antibodies to the challenge virus were detected in most pigs (7/12) and virus neutralizing antibodies in all pigs. There was no antigen-dose dependent effect on the antibody response among the pigs immunized with adjuvanted H5N1 vaccines. After challenge, these pigs showed a complete clinical protection, reduced lung lesions and a significant protection against virus replication in the respiratory tract. Though the challenge virus showed only moderate replication efficiency in pigs, our study suggests that AS03(A)-adjuvanted H5N1 vaccine may confer a broader protection than generally assumed. The pros and cons of the pig as an H5N1 challenge model are also discussed.

Evaluation of systemic and mucosal anti-HPV16 and anti-HPV18 antibody responses from vaccinated women.

Ideal methods to monitor HPV neutralizing antibodies induced by vaccination have not been established yet. Here, we evaluated systemic and cervical antibody levels induced by HPV16/18 AS04-adjuvanted vaccine (GlaxoSmithKline Biologicals) using a secreted alkaline phosphatase neutralization assay (SEAP-NA) and enzyme-linked immunosorbent assay (ELISA). Serum and cervical secretions from 50 vaccinated women were used to assess (1) overall assay reproducibility; (2) inter-assay and inter-specimen correlation; (3) correlations between month 1 and month 12 titers. Strong correlations between SEAP-NA and ELISA were observed (serum anti-HPV16/18, rho=0.91/0.85; cervix anti-HPV16/18, rho=0.84/0.89). Systemic and cervical antibody measures also correlated well (rho range: 0.64-0.75); except at mid-cycle (rho range: 0.28-0.65). Correlations between antibody levels at 1 and 12 months following the start of vaccination were poor (rho range: 0.16-0.38). In conclusion, HPV16/18 VLP-based ELISA is a reliable and valid method to monitor anti-HPV16/18 neutralizing potential for the first year following vaccination; however, additional studies will be required to better define the effects of the time on cycle and patterns of antibody response over time following vaccination.

Evidence for physical interaction between the immunoglobulin heavy chain variable region and the 3' regulatory region.

B cell-specific expression of immunoglobulin heavy chain (IgH) genes utilizes two cis regulatory regions, the intronic enhancer (Emicro), located in the J(H)-Cmicro intron, and a complex regulatory region that lies 3' to the IgH gene cluster, 3' RR. We hypothesized that the 3' RR is involved in IgH gene transcription in plasma cells via physical interaction between distal 3' RR enhancers and target V(H) sequences, with loop formation by intervening DNA. In support of this hypothesis we report sequence data at DNA recombination breakpoints as evidence for loop formation preceding DNA inversion in a plasma cell line. In addition, using the chromosome conformation capture technique, physical interactions between V(H) and 3' RR were analyzed directly and detected in MPC11 plasma cells and variants and normal splenic B cells but not detected in splenic T cells or in non-B cells. V(H)-3' RR interactions were present in the absence of Emicro, but when the hs1,2 enhancer was replaced by a Neo(R) gene in a variant cell line lacking Emicro, H chain expression was lost, and interactions between V(H) and 3' RR and among the 3' RR regulators themselves were severely disrupted. In addition, the chromosome conformation capture technique detected interactions between the myc promoter and 3' RR elements in MPC11, which like other plasmacytomas contains a reciprocal translocation between the c-myc and the IgH locus. In sum, our data support a hypothesis that cis V(H)-3' RR and myc-3' RR interactions involve physical interactions between these DNA elements.

Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only.

An effective virus-like particle (VLP) based prophylactic vaccine designed to protect against persistent infection with human papillomavirus (HPV) types 16 and 18 and subsequent lesion development will need to induce a strong humoral and cellular immune response capable of providing long-term protection. Our objective was to evaluate the ability of an HPV16/18 L1 VLP vaccine formulated with the AS04 adjuvant system (3-O-desacyl-4'-monophosphoryl lipid A (MPL) and aluminium salt) to induce an immune response of higher magnitude and persistence compared to a vaccine formulated with aluminium salt only. We demonstrated that MPL adsorbed onto aluminium salt retains its capacity to activate an innate immune response as assessed by the production of TNFalpha by human monocytes (U937). In addition, vaccination of mice, monkeys or human subjects with AS04 formulations induced higher total anti-L1 VLP16 and L1 VLP18 antibody responses (1.6-8.5-fold) than the aluminium salt only formulations. The enhanced antibody response induced by the AS04 vaccine formulation (1.6-4.1-fold) in monkeys and humans was shown to be targeted to functional neutralising L1 VLP16 and L1 VLP18 epitopes as assessed by V5/J4 specific ELISAs or HPV16 and HPV18 pseudo-neutralization assays. The enhanced immune profile observed with the AS04 formulation in terms of both total, V5/J4 specific and neutralizing antibodies was shown to persist for at least 3.5-year post-vaccination in human subjects. Finally, using the newly developed B cell ELISPOT assay we also demonstrated that the AS04 formulation elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminium salt only formulations. These data strongly support the role of the AS04 adjuvant, which includes the immunostimulant MPL, in triggering a persistent vaccine-induced immune response of high quality.

Influence of the mucosal epithelium microenvironment on Langerhans cells: implications for the development of squamous intraepithelial lesions of the cervix.

We have addressed the notion that the initiation and progression of human papillomavirus associated cancer of the uterine cervix are associated with alterations of Langerhans cells (LC) within the mucosal squamous epithelium. Since the transformation zone (TZ) of the cervix is the site where the majority of squamous intraepithelial lesions (SIL) are initiated, in contrast to the exocervix, we decided to investigate the influence of the local microenvironment within the TZ on the function and density of LC. We show that the TZ is associated with a significant reduction in the density of immature LC (CD1a/LAG) compared to the exocervix. In contrast, the development of SILs is attributed with a relative increased density of immature LC, compared to the TZ. Furthermore, we show that this variability in LC density is correlated with a differential expression of TNFalpha and MIP3alpha within the micro-environment of the TZ and SILs. Both TZ and SIL epithelium-derived LC, in the presence of allogeneic PBMC, induced lower levels of proliferation and IL2 production and higher levels of the immunosuppressive cytokine IL10 in comparison to the exocervix. Nevertheless, the epithelium-derived LC in SILs exhibits a reduction in their functional activity, relative to the TZ. Together our studies suggest that the immunosurveillance within the epithelium of the TZ may be intrinsically perturbed due to the altered expression of chemokines/cytokines and the concomitant diminished density of LC. Furthermore, following HPV infection and the development of SILs, the function of LC may be further incapacitated by viral associated mechanisms.