Germain J. P. Fernando

Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy.

Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins

Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy.

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1‐ and Th2‐type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H‐2b‐restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H‐2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL‐2 and IL‐4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide‐pulsed, and whole E7 gene‐transfected tumour target cells; and (iv) in vivo protection against an E7 gene‐transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour‐associated antigens) without the unwanted side effects of oil‐based adjuvants. In addition they support the case for a E7‐targeted therapeutic vaccine for HPV‐associated human cervical cancer.

The number of long-lasting functional memory CD8+ T cells generated depends on the nature of the initial nonspecific stimulation

The mechanism of generation of memory cytotoxic T cells (CTL) following immunization remains controversial. Using tumor protection and IFN‐γ ELISPOT assays in mice to detect functional CTL, we show that the initial effector CTL burst size after immunization is not directly related to the amount of functional memory CTL formed, suggesting that memory CTL are unlikely to arise stochastically from effector CTL. Induction of MHC class II‐restricted T helper cells at the time of immunization by inclusion of a T helper peptide or protein in the immunogen, is necessary to generate memory CTL, although no T helper cell induction is required to generate effector CTL to a strong MHC class I‐binding peptide. Host protective T cell memory correlates with the number of CTL epitope responsive IFN‐γ‐secreting memory T cells as measured in an ELISPOT assay at the time of tumor challenge. We conclude that a different antigen presenting environment is required to induce long‐lasting functional memory CTL, and non‐cognate stimulation of the immune system is essential to allow generation of a long‐lasting host protective memory CTL response.

The vaccinia virus K2L gene encodes a serine protease inhibitor which inhibits cell-cell fusion

In certain circumstances, cells infected with vaccinia virus (VV) undergo fusion, but this does not occur in tissue cultures infected with wild-type VV. The VV genome includes three genes (B24R, B13R, and K2L) encoding polypeptides that are structurally related to members of the plasma serine proteases inhibitor (SPI) superfamily. In this study, we demonstrate by deleting these genes singly or in combination that the K2L gene encoding SPI-3, but not the B24R or B13R genes encoding SPI-1 and SPI-2, inhibits cell-cell fusion in VV-infected cells. A VV-encoded hemagglutinin (HA) has previously been demonstrated to inhibit cell-cell fusion, but fusion-promoting VVs with K2L gene deletions had normal expression and cellular location of the VV HA. As both HA and SPI-3 independently inhibit cell-cell fusion in VV-infected cells, there must be at least two fusion-promoting mechanisms encoded by VV. These may play different roles in virus-cell fusion and in cell-cell fusion after VV infection.

IL-10 Mediates Suppression of the CD8 T Cell IFN-γ. Response to a Novel Viral Epitope in a Primed Host

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-γ effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as priming to virus without CTL induction is associated with inhibition. However, IL-10−/− mice, in contrast to IL-10+/+ mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-γ-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.

Cell-mediated immune responses to E7 peptides of human papillomavirus (HPV) type 16 are dependent on the HPV type infecting the cervix whereas serological reactivity is not type-specific

Forty-two women attending a colposcopy clinic for evaluation of abnormal cervical cytology and 13 normal controls were studied for the presence of lymphocyte proliferation (LP) cell-mediated immune (CMI) responses and serological reactivity to E7 peptides of human papillomavirus type 16 (HPV-16). HPV was typed by Southern blot hybridization of exfoliated cervicovaginal cell DNA. Positive LP responses (stimulation index > or = 5.0) to one or more E7 peptides were observed in 28.6% (12 of 42) of patients and 23.1% (three of 13) of controls. Of patients infected with HPV-16, -31 or -33, 63.6% (seven of 11) showed a positive LP response compared with 14.3% (two of 14) of women infected with other HPV types (P = 0.02), 17.6% (three of 17) negative for HPV (P = 0.02) and 23.1% (three of 13) of controls (HPV status unknown) (P = 0.05). C-terminal peptide 109 (amino acids 72 to 97) elicited positive LP responses in 45.4% (five of 11) of patients infected with HPV -16, -31 or -33 compared with 7.1% (one of 14) patients infected with other HPVs (P = 0.04), 5.9% (one of 17) of women negative for HPV (P = 0.02) and 7.7% (one of 13) of controls (P = 0.05). HPV-16 group-specific LP responses of borderline significance were also observed against E7 peptides 103, 105 and 108 (17-37, 37-54 and 62-80) (P = 0.07). ELISA reactivity (IgG) to E7 peptide 109 (72-97) was present in 7.7% (one of 13) of controls, 35.3% (six of 17) of HPV-negative patients, 42.9% (six of 14) of patients infected with other HPVs, and only 9.1% (one of 11) of patients infected with HPV-16, -31 or -33. CMI responses to C-terminal HPV-16 E7 peptide 109 (72-97) were thus significantly related to ongoing cervical infection with HPV-16 and closely related types, whereas serological reactivity to E7 peptides was not HPV type-specific.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II‐deficient mouse, I‐Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I‐Ab‐restricted CD4+ T cells (Laufer, T. M. et al., Nature 1996. 383:81u2009–u200985). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7‐transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H‐2q) or F1(C57BL/6Ju2009×u2009FVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7‐transgenic and non‐transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non‐transgenic animals. Further, E7‐immunized non‐transgenic F1 (FVBu2009×u2009C57BL/6J) animals developed strong E7‐specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7+ tumors, whereas similarly immunized K14E7‐transgenic animals had a markedly reduced CTL response to E7 and no E7‐specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7‐specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7‐specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in “split” tolerance to that autoantigen.

Peptide polymerisation facilitates incorporation into ISCOMs and increases antigen-specific IgG2a production.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.

Overcoming Original Antigenic Sin to Generate New CD8 T Cell IFN-γ Responses in an Antigen-Experienced Host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-γ CD8+ T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4+ T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-γ production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-γ response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.