To elucidate the role of innate responses in vaccine immunogenicity, we compared early responses to hepatitis B virus (HBV) surface antigen (HBsAg) combined with different Adjuvant Systems (AS) in healthy HBV-naïve adults, and included these parameters in multi-parametric models of adaptive responses. A total of 291 participants aged 18–45 years were randomized 1:1:1:1:1 to receive HBsAg with AS01B, AS01E, AS03, AS04, or Alum/Al(OH)3 at days 0 and 30 (: NCT00805389). Blood protein, cellular, and mRNA innate responses were assessed at early time-points and up to 7 days after vaccination, and used with reactogenicity symptoms in linear regression analyses evaluating their correlation with HBs-specific CD4+ T-cell and antibody responses at day 44. All AS induced transient innate responses, including interleukin (IL)-6 and C-reactive protein (CRP), mostly peaking at 24 h post-vaccination and subsiding to baseline within 1–3 days. After the second but not the first injection, median interferon (IFN)-γ levels were increased in the AS01B group, and IFN-γ-inducible protein-10 levels and IFN-inducible genes upregulated in the AS01 and AS03 groups. No distinct marker or signature was specific to one particular AS. Innate profiles were comparable between AS01B, AS01E, and AS03 groups, and between AS04 and Alum groups. AS group rankings within adaptive and innate response levels and reactogenicity prevalence were similar (AS01B ≥ AS01E > AS03 > AS04 > Alum), suggesting an association between magnitudes of inflammatory and vaccine responses. Modeling revealed associations between adaptive responses and specific traits of the innate response post-dose 2 (activation of the IFN-signaling pathway, CRP and IL-6 responses). In conclusion, the ability of AS01 and AS03 to enhance adaptive responses to co-administered HBsAg is likely linked to their capacity to activate innate immunity, particularly the IFN-signaling pathway.

The human papillomavirus (HPV) E2 protein plays an important role in viral DNA replication. Many studies with high-risk HPVs have demonstrated that the E2 protein can also repress transcription of the E6 and E7 oncogenes. This conclusion, based on experiments carried out with cervical cancer cells bearing integrated HPV genomes, is currently assumed to be applicable to the normal HPV life cycle, in which the viral genomes are episomal. Here, we have tested experimentally whether this assumption is correct. We made use of a pair of isogenic cell lines, W12 and S12. W12 cells contain episomal HPV16 genomes, whereas S12 cells, which are derived from the W12 line, contain HPV DNA as integrated copies. When we expressed E2 in S12 cells, we observed strong repression of E6 and E7 transcription. In contrast, no effect of E2 on the transcription of these genes was detected in W12 cells. While integration of the viral genome into the host DNA contributes to the difference between W12 and S12 cells, integration by itself is not sufficient to explain this difference. Instead, the chromatin structure in the region of the E6 and E7 promoter (p97), which we show to be very different in these two cell lines, is likely to be the cause of the different responsiveness of p97 to the E2 protein. Experiments with the histone deacetylase inhibitor trichostatin A (TSA) indicated that the episomal HPV16 DNA is in a relatively inaccessible state prior to TSA treatment. Our results, together with those of others, suggest that any effect of the E2 protein on the expression of the E6 and E7 genes during the normal viral life cycle is of secondary importance compared to the function of E2 in replication.Cervical cancer is one of the leading causes of death from cancer among women worldwide. The principal factor responsible for this cancer is infection of the cervical epithelium by some types of human papillomaviruses (HPVs). The HPV capsid proteins enclose the viral minichromosome, which is composed of approximately 8,000 bp of circular double-stranded DNA, in the form of nucleosomes similar to those of cellular chromatin. The HPV type 16 (HPV16) DNA encodes eight proteins required for viral DNA replication, encapsidation, and virus release. In addition, there is a noncoding region called the long control region (LCR), which contains enhancer elements and a promoter (p97 in HPV16 and p105 in HPV18) from which the viral oncogenes E6 and E7 are transcribed. The E7 protein binds and inactivates the pRb tumor suppressor protein and in some instances targets its destruction (16,20,32,33). This results in the release of the E2F transcription factor and the consequent expression of many S-phase genes whose promoters are responsive to E2F. The E6 protein exhibits many different effects in the cell, of which destruction of the p53 tumor suppressor protein (32,39,40) and the activation of telomerase (28, 29) are two that contribute to tumorigenesis. Overexpression of the E6 and E7 proteins is thought to be responsible for initiating and maintaining p...

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed.The host tissue of human papillomaviruses is the stratified epithelium. This tissue is complex in that it is composed of layered sheets of nondividing cells in various stages of terminal differentiation, with the uppermost layer being the most differentiated. Only cells of the bottom-most layer of this tissue, the basal cells, proliferate. Although the HPV life cycle begins with the infection of a basal cell, it only comes to completion when the infected cell reaches the upper layers of the epithelium. As a consequence, the HPV DNA initially finds itself in the nucleus of a proliferating cell, but later in that of a differentiating (nonproliferating) one. This sequential mixture of cell states that the virus has to contend with has undoubtedly shaped the way by which HPV replicates its DNA throughout the varying milieux during its life cycle.It is proposed that immediately after infection, the papillomavirus DNA copy number is amplified to a certain level (50 to 400) per cell (9). This first amplification replication is believed to be rapid and transient, after which the viral DNA is stably maintained at this level in subsequent divisions of the basal cell. This is thought to be achieved by maintenance replication, where the viral episomes approximately double their copy number during the S phase of the host cell cycle and segregate to the resulting two daughter cells. After differentiation of the host cell, the viral DNA undergoes another amplification step, the second amplification replication, which increases the HPV DNA copy number to several hundreds or thousands per cell. This is followed by packaging of the viral DNA into virus particles. Although this triphasic model has not been proven in its entirety, it is supported by va...

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immunogenicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis B surface-specific antibody response and was characterized by positive regulation of genes associated with interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell–associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination. Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature across individuals.