Jerry L. Blackwell

Evidence for Potent Autologous Neutralizing Antibody Titers and Compact Envelopes in Early Infection with Subtype C Human Immunodeficiency Virus Type 1

ABSTRACT Information about neutralizing antibody responses in subtype C-infected individuals is limited, even though this viral subtype causes the majority of AIDS cases worldwide. Here we compared the course and magnitude of the autologous neutralizing antibody (NAb) response against viral envelope (Env) glycoproteins present during acute and early infection with subtypes B and C human immunodeficiency virus type 1 (HIV-1). NAb responses were evaluated in 6 subtype B-infected and 11 subtype C-infected subjects over a mean evaluation period of 25 months using a pseudovirus reporter gene assay. All subjects in the C cohort were infected through heterosexual contact, while five of the six subjects in the B cohort were infected via male-to-male contact. The kinetics and magnitude of the NAb responses varied among subjects in the B and C cohorts; however, the median 50% inhibitory concentration (IC50 titer) reached by antibody in the plasma of subtype C-infected subjects, overall, was 3.5-fold higher than in the subtype B-infected subjects (P = 0.06). The higher titers of NAbs in the C cohort were associated with viruses having significantly shorter amino acid length (P = 0.002) in the V1 to V4 region of the surface Env glycoprotein, gp120, compared to the B cohort. Despite the potency of the autologous subtype C NAb response, it was not directed against cross-neutralizing epitopes. These data demonstrate that subtype C Envs elicit a potent yet restricted NAb response early in infection that frequently reaches IC50 titers in excess of 1:1,000 and suggest that clade-specific differences may exist in Env immunogenicity or susceptibility to neutralization.

Role of V1V2 and Other Human Immunodeficiency Virus Type 1 Envelope Domains in Resistance to Autologous Neutralization during Clade C Infection

ABSTRACT Biologically functional clade C envelope (Env) glycoproteins from the chronically (donor) and newly (recipient) infected partners of four heterosexual transmission pairs in Zambia were cloned and characterized previously. In each case, the donor viral quasispecies contained Envs that were resistant to autologous neutralization by contemporaneous plasma, while the recipient Envs were sensitive to neutralizing antibodies in this donor plasma sample. The donor Envs also varied in length, glycosylation, and amino acid sequence of the V1V2 hypervariable domain of gp120, while the recipient Envs were much more homogeneous. To assess the contribution of V1V2 to the neutralization phenotype of the donor Envs, V1V2 domains from neutralization-sensitive recipient Envs were replaced with donor V1V2 domains, and the autologous neutralization sensitivities of the chimeric Envs were evaluated using a virus-pseudotyping assay. Long donor V1V2 domains regulated sensitivity to autologous neutralization, although the effect was dependent on the Env background. Short donor V1V2 domains did not confer neutralization resistance. Primary sequence differences in V2 were also found to influence neutralization sensitivity in one set of recipient Envs. The results demonstrate that expansion of the V1V2 domain is one pathway to escape from autologous neutralization in subtype C Envs. However, V1V2-independent mechanisms of resistance also exist, suggesting that escape is multifaceted in chronic subtype C infection.

Interaction between the Cellular Protein eEF1A and the 3′-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis

ABSTRACT RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3′(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3′(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3′ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3′ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3′ SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3′ end of the genome and the RC. eEF1A bound with similar efficiencies to the 3′-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Adenoviral Gene Therapy for Renal Cancer Requires Retargeting to Alternative Cellular Receptors

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for cancer. However, a primary limiting factor for the use of Ad vectors for cancer gene therapy is their critical dependence on cellular expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. Following the identification of CAR deficiency in RCC lines, we have found abundant membrane expression of alpha(v)beta 3 and alpha(v)beta 5 integrins and of the putative receptor to Ad serotype 3 (Ad3). As an alternative gene therapy approach for RCC that would circumvent CAR deficiency, we employed retargeting of replication-incompetent Ad vectors and replication-competent Ad viruses to alpha(v)beta 3 and alpha(v)beta 5 integrins and to the putative Ad3 receptor. These strategies to genetically alter Ad tropism were based on either the insertion of a cysteine-aspartate-cysteine-arginine-glycine-aspartate-cysteine-phenylalanine-cysteine (RGD) motif into the HI loop of the Ad fiber knob domain or on generation of a chimeric Ad fiber composed of adenovirus serotype 5 shaft/Ad3 knob. Both strategies proved highly efficient to circumvent CAR deficiency and enhance gene delivery into RCC cells. Furthermore, in the context of replication-competent Ad, tropism alteration resulted in distinct capacity of the retargeted viruses to infect, replicate, and lyse RCC models in vitro and in vivo. The retargeting strategies were particularly beneficial in the context of replication-competent Ad. These findings underscore the importance of CAR-independent cellular entry mechanisms in RCC and are highly consequential for the development of viral antitumor agents for RCC and other CAR-negative tumors.

XMRV infection in patients with prostate cancer: novel serologic assay and correlation with PCR and FISH.

OBJECTIVES To develop a serum-based assay to detect neutralizing antibodies to the xenotropic murine leukemia virus-related virus (XMRV) retrovirus and to use this assay with polymerase chain reaction and fluorescence in situ hybridization to identify patients with prostate cancer previously exposed to XMRV infection and those who carry XMRV viral sequences in their prostate. METHODS Patients who had undergone radical prostatectomy were enrolled, and biologic specimens were obtained at surgery. The patients were genotyped for the R462Q RNASEL variant using a TaqMan genotyping assay on DNA from the peripheral blood. A serum assay that detects XMRV neutralizing antibodies was developed and used to determine which patients had serologic evidence of previous infection with XMRV virus. Some of these patients were also tested for the presence of XMRV nucleotide sequences in their prostate using polymerase chain reaction and fluorescence in situ hybridization analysis. RESULTS At a serum dilution of 1:150, our assay detected 11 (27.5%) of 40 patients with XMRV neutralizing antibodies, including 8 (40%) of 20 with the RNASEL genotype QQ and 3 (15%) of 20 with either the RQ or RR genotype. These results were in complete concordance with 2 other assays (polymerase chain reaction and fluorescence in situ hybridization), which were designed to detect XMRV infection. CONCLUSIONS XMRV infects some patients with prostate cancer. Neutralizing antibodies against XMRV correlated with 2 independent methods of detecting the virus in the prostate. The antibody response suggests that with clinical serologic assay development, it might be possible to screen patients for XMRV infection. The cases presented in the present report provided biologic samples that can be used for the development of a clinically relevant assay.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both αv integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

Unique Mutational Patterns in the Envelope α2 Amphipathic Helix and Acquisition of Length in gp120 Hypervariable Domains Are Associated with Resistance to Autologous Neutralization of Subtype C Human Immunodeficiency Virus Type 1

ABSTRACT Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendalls tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donors antibodies, and five of these were located within an 18-residue amphipathic helix (α2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire α2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in α2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the α2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the α2 helix for efficient viral replication and immune avoidance.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma

Midkine (MK), a heparin binding growth factor, and cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandin, are both up-regulated at the mRNA or protein level in many human malignant tumors. Here, we investigated the tumor specificity of both MK and COX-2 promoters in human pancreatic cancer, with the aim to improve the selectivity of therapeutic gene expression. We constructed recombinant adenoviral (Ad) vectors containing either the luciferase (Luc) reporter gene under the control of the COX-2 or MK promoter or the herpes simplex virus thymidine kinase (HSV Tk) gene under the control of the COX-2 promoter and compared the expression with the cytomegalovirus (CMV) promoter. AdMKLuc achieved moderate to relatively high activity upon infection to both primary and established pancreatic carcinoma cells. Of the two COX-2 promoter regions (COX-2M and COX-2L), both revealed a high activity in primary pancreatic carcinoma cells, whereas in the established pancreatic carcinoma cell lines, COX-2L has an approximately equal high activity compared to CMV. In addition, both AdCOX-2M Tk and AdCOX-2L Tk induced marked cell death in response to ganciclovir (GCV) in three of four established pancreatic carcinoma cell lines. From these results, and because it has been reported that AdMKTk and AdCOX-2L Tk in combination with GCV did not reveal significant liver toxicity, we conclude that the MK as well as the COX-2 promoters are promising tumor-specific promoters for Ad vector–based gene therapy of pancreatic cancer. Cancer Gene Therapy (2001) 8, 990–996

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.