A novel human papillomavirus type 6 neutralizing domain comprising two discrete regions of the major capsid protein L1.

We have mapped the binding sites on human papillomavirus (HPV) type 6 for three HPV 6-specific neutralizing monoclonal antibodies (mAbs). The critical binding residues were first identified by making HPV 11-like amino acid substitutions in the HPV 6 major capsid protein L1 and assaying the resulting virus-like particles (VLPs) for reactivity with the mAbs. To confirm the relevance of these residues for mAb binding, we demonstrated that HPV 6 type-specificity could be transferred to HPV 11 VLPs by making the appropriate HPV 6-like amino acid substitutions in the HPV 11 L1. Two binding regions were found. For one mAb, all critical residues are centered at residue 53, while for the other two mAbs, type-specific binding also requires a second site located more than 100 residues distal to the first. Both binding sites coincide with regions of L1 where the sequences of the closely related HPV 6 and 11 diverge. These regions are where the L1 sequences are the least well conserved among all HPV types and they have been implicated in type-specific binding for other HPV types. This suggests that clusters of diverged residues, surrounded by conserved L1 sequences, are presented on the surface of assembled particles and are responsible for eliciting critical humoral immune responses to the virus.

A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay.

In this study, we describe a simple and efficient method for both the monitoring of antigen-specific CD4 and CD8 T cell responses as well as the identification of novel CD4 and CD8 T cell epitopes using a modified ELISpot assay and pools of 20mer peptides. We have demonstrated that pools containing as many as 64 20mer peptides may be used to screen for CD4 and CD8 T cell responses to HPV16 L1, E1, and E7 in mice. Using arrays of pools of overlapping 20mer peptides, we have identified novel CD4 and CD8 epitopes in both HPV16L1 and HPV16E1 which are presented in Balb/c mice. We have further shown that the use of 20mer peptides is equivalent to using minimal 9mer epitopes for the stimulation of CD8 T cell responses in our assay. While our experiments are conducted in mice, the use of peptide pool arrays allows for the identification of epitope-specific responses using far fewer cells than is required for testing a panel of overlapping peptides individually, making this strategy particularly useful in clinical settings where immune cells may be limiting.

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.

HPV11 mutant virus-like particles elicit immune responses that neutralize virus and delineate a novel neutralizing domain.

Characterization of the regions of human papillomaviruses (HPVs) that elicit neutralizing immune responses supports studies on viral infectivity and provides insight for the development and evaluation of prophylactic vaccines. HPV11 is a major etiologic agent of genital warts and a likely vaccine candidate. A conformationally dependent epitope for the binding of three neutralizing monoclonal antibodies (mAbs) has been mapped to residues G(131)T(132) of the L1 major capsid protein. The mAbs bind L1 only when it is assembled into virions or into virus-like particles (VLPs) that mimic the capsid structure. We were interested in identifying other domains of L1 that elicit neutralizing responses. To this end, we have generated a panel of mAbs against VLPs derived from HPV11 L1 harboring a G131S substitution. The new mAbs are unlike the neutralizing mAbs previously mapped to residues G(131)T(132) in that they bind both prototype and HPV11:G131S mutant VLPs. Some of the new mAbs neutralized virus in vitro. We have mapped epitopes for three of these new mAbs, as well as a neutralizing mAb generated against HPV11 virions, by measuring binding to HPV6 VLPs substituted with HPV11-like amino acids. Two regions are critical: one defined by HPV11 L1 residues 263-290 and the other by residues 346-349. mAbs H11.H3 and H11.G131S.G3 bind HPV6 VLPs with substitutions derived from the 346-349 region; in addition, H11.G131S.G3 binds HPV6 VLPs with substitutions derived only from the 263-290 region. Although H11.H3 does not bind HPV6 VLPs with substitutions derived from the 263-290 region, binding to HPV6 VLPs is enhanced when both sets of substitutions are present. mAbs H11.G131S.I1 and H11.G131S.K5 bind HPV6 VLPs with the 263-290 substitutions, but show little binding to HPV6 VLPs with the 346-349 substitutions. However, binding to HPV6 VLPs is enhanced when substitutions at both regions are present. The 346-349 region has not previously been described as eliciting a neutralizing response for any HPV type. In addition, the work demonstrates a complex binding site contributed by two distinct regions of L1.

Existence and learning of oscillations in recurrent neural networks.

In this paper we study a particular class of -node recurrent neural networks (RNN's). In the 3-node case we use monotone dynamical systems theory to show, for a well-defined set of parameters, that, generically, every orbit of the RNN is asymptotic to a periodic orbit. Then we investigate whether RNN's of this class can adapt their internal parameters so as to "learn" and then replicate autonomously (in feedback) certain external periodic signals. Our learning algorithm is similar to identification algorithms in adaptive control theory. The main feature of the algorithm is that global exponential convergence of parameters is guaranteed. We also obtain partial convergence results in the -node case.

Enhancement of DNA vaccine potency using conventional aluminum adjuvants.

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5-10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

The prophylactic effect of immunization with DNA encoding herpes simplex virus glycoproteins on HSV-induced disease in guinea pigs.

Plasmid expression vectors encoding herpes simplex virus type 2 (HSV-2) glycoproteins B (gB) or D (gD) were constructed and tested for their ability to immunize guinea pigs against genital HSV infection. Immunization with a plasmid expressing the aminoterminal 707 amino acids (aa) of gB induced humoral immune responses detected by ELISA and virus neutralization. When challenged by vaginal infection, immunized animals were partially protected from genital herpes, exhibiting significantly reduced primary and subsequent recurrent disease. When the gB plasmid was combined with a plasmid expressing full-length gD, immunized guinea pigs developed humoral responses to both proteins and were also significantly protected from viral challenge.

Immunization of non-human primates with DNA vaccines.

The pre-clinical efficacy of DNA vaccines has been demonstrated in a number of animal models, but more limited data exist regarding their immunogenicity in non-human primates. The studies described below demonstrate that DNA vaccines in reasonable dosages encoding a variety of viral proteins could result in the generation of antibodies, neutralizing antibodies, or cytotoxic T lymphocytes in primates. Furthermore, these responses could be boosted by repeat administration of the DNA vaccine. In an effort to assess the safety of such vaccines sera from primates was shown to lack anti-DNA antibodies.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds.

Synthesis of influenza virus mRNA is primed by capped and methylated (cap 1, m7GpppXm) RNAs which the virus derives by endonucleolytic cleavage from RNA polymerase II transcripts in host cells. The conserved nature of the endonucleolytic processing provides a unique target for the development of antiviral agents for influenza viruses. A series of 4-substituted 2,4-dioxobutanoic acid compounds has been identified as selective inhibitors of this activity in both influenza A and B viruses. These inhibitors exhibited 50% inhibitory concentrations in the range of 0.2 to 29.0 microM for cap-dependent influenza virus transcription and had no effect on the activity of other viral and cellular polymerases when tested at 100- to 500-fold higher concentrations. The compounds did not inhibit the initiation or elongation of influenza virus mRNA synthesis but specifically inhibited the cleavage of capped RNAs by the influenza virus endonuclease and were not inhibitory to the activities of other nucleases. Additionally, the compounds specifically inhibited replication of influenza A and B viruses in cell culture with potencies comparable to the 50% inhibitory concentrations obtained for transcription.

The DNA binding domain of herpes simplex virus type 1 origin binding protein is a transdominant inhibitor of virus replication.

The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.

Cooperative interactions between replication origin-bound molecules of herpes simplex virus origin-binding protein are mediated via the amino terminus of the protein.

The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.

Oligopeptides inhibit the ribonucleotide reductase of herpes simplex virus by causing subunit separation.

It has recently been reported that a nonapeptide (H-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH) identical in sequence to the last nine amino acid residues of RR2, the small subunit of herpes simplex virus (HSV) ribonucleotide reductase (E.C. 1.17.4.1; RR), can inhibit HSV RR activity in vitro. It has been postulated that this peptide acts by interfering with the interaction of the large subunit, RR1, and RR2, but direct demonstration of its mechanism of action has not been reported. We present data which show that the inhibition of HSV RR by this oligopeptide is due to induced subunit dissociation.

Activation of the transforming potential of a normal cell sequence: a molecular model for oncogenesis.

The molecularly cloned, long terminal repeat (LTR) of the Moloney sarcoma virus (M-MSV) provirus has been covalently linked to c-mos, the cellular homolog of the M-MSV-specific sequence, v-mos. These newly constructed clones lack any M-MSV-derived sequences other than the LTR, but in DNA transfection assays they transform cells as efficiently as cloned subgenomic M-MSV fragments containing both v-mos and LTR. Cells transformed by LTR:c-mos hybrid molecules contain additional copies of mos DNA, and several size classes of polyadenylated RNA's with sequence homology to mos. The activation of the transforming potential of c-mos by the proviral LTR suggests a model whereby LTR-like elements could activate other normal cell sequences with oncogenic potential.

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.

Nucleotide sequences of integrated Moloney sarcoma provirus long terminal repeats and their host and viral junctions.

Integrated Moloney murine sarcoma provirus (MSV) has direct terminal repeat sequences (TRS). We determined the nucleotide sequence of both 588-base-pair TRS elements and the adjacent host and viral junctions of an integrated MSV cloned in bacteriophage lambda. Sequences were identified corresponding to the tRNAPro primer binding site in genomic RNA and the reverse-transcribed minus strong stop DNA. Each 588-base-pair repeat contains putative sites for promoting RNA synthesis and RNA polyadenylylation. The first and last 11 nucleotides of the TRS are inverted with respect to each other, and the same four-nucleotide host sequence is found bracketing integrated MSV. Some similarities of TRS and prokaryotic insertion sequence elements are discussed.

Biological activity of cloned Moloney sarcoma virus DNA: Terminally redundant sequences may enhance transformation efficiency.

We have measured the ability of cloned restriction fragments containing the whole and partial genomes of two strains of Moloney murine sarcoma virus to induce cell transformation in DNA transfection assays. The cloned intact ml and HTl murine sarcoma virus proviruses transform with an efficiency of approximately 40,000-50,000 focus-forming units/pmol of proviral DNA, and the majority of these transformed cells contain a rescuable viral genome. A cloned 2.1-kilobase-pair internal fragment of the murine sarcoma virus containing 1.2 kilobase pairs of sarcoma virus-specific sequences (src) and approximately 900 base pairs of leukemia virus-derived sequences adjacent to the 5' end of src transforms with approximately 1/10,000th the efficiency of the intact genome. When leukemia virus-deprived sequences containing a single copy of the 600-base-pair direct terminal repeated sequences are present at either the 5' or 3' end of this src-containing fragment, the transforming activity is stimulated 1000-fold. Cotransfection with a mixture of cloned fragments, one containing the internal 2.1-kilobase-pair src fragment and the other containing a single copy of the terminally redundant sequence, results in a 300-fold increase in transformation efficiency.