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. 2003 Mar;77(5):3119-30.
doi: 10.1128/jvi.77.5.3119-3130.2003.

Induction of primary virus-cross-reactive human immunodeficiency virus type 1-neutralizing antibodies in small animals by using an alphavirus-derived in vivo expression system

Ming Dong  1 , Peng Fei Zhang, Franziska Grieder, James Lee, Govindaraj Krishnamurthy, Thomas VanCott, Christopher Broder, Victoria R Polonis, Xiao-Fang Yu, Yiming Shao, Dennis Faix, Patricia Valente, Gerald V Quinnan Jr
Affiliations

Induction of primary virus-cross-reactive human immunodeficiency virus type 1-neutralizing antibodies in small animals by using an alphavirus-derived in vivo expression system

Ming Dong et al. J Virol. 2003 Mar.

Abstract

We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160deltaCT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160deltaCT was released by cells in the form of membrane-bound vesicles. gp160deltaCT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virus-neutralizing activity by vaccination may be realized.

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Figures

FIG. 1.
FIG. 1.
Expression and processing of the HIV-1 envelope by VEE replicons. (A) Protein produced by cells transfected with replicon transcripts expressing R2 gp140. Pooled fractions obtained by size exclusion chromatography containing predominantly either gp120 or gp140 were cross-linked with 100 mM EGS and analyzed by Western blotting as described in Materials and Methods. (B) BHK cells were inoculated with replicon particle preparations expressing R2 gp140, gp160, or gp160ΔCT and then assayed by immunofluorescence for HIV-1 envelope expression. Sites of gp160ΔCT antigen capping (C) and vesicle formation (V) are shown in subpanels I, II, and III.
FIG. 2.
FIG. 2.
Envelope protein binding antibodies and neutralizing activity in sera from BALB/c mice immunized by inoculation with VEE replicon particles expressing HIV-1 gp140 or gp160. Mice were injected in the footpad with replicon particle suspensions at the times indicated by the vertical arrowheads. Blood for assays was collected approximately 2 weeks after each immunization. Neutralization assays were performed with pseudotyped viruses. Neutralization titers were determined to be the highest immune serum dilutions at which >50% inhibition of luciferase activity was obtained, compared to nonimmune serum. In experiment I, mice were immunized repeatedly with either 5 × 106 FFU (closed circles) or 105 FFU (open circles) of replicon particles expressing gp140. In experiment II, mice were immunized with gp160-expressing replicons throughout (circles), gp140-expressing replicons throughout (squares), or gp160-expressing replicons for two doses, followed by gp140-expressing replicons for subsequent doses (triangles).
FIG. 3.
FIG. 3.
Influence of VEE envelope protein host range mutations on immunogenicity of VEE replicons expressing HIV-1 envelope proteins. C3H/He mice were immunized by footpad inoculation at the times indicated by the vertical arrows. In panel A, EIA titers are presented as means ± standard deviations for groups of six mice each immunized with 5 × 106 FFU of pGP-gp140 per dose (⧫) or 5 × 105 FFU of pGPm-gp140 per dose (•). In panel B, the heights of the columns indicate the geometric mean neutralization titers of sera from mice immunized with either pGP-gp140- or pGPm-gp140-expressing replicons against strain SF162 pseudotyped virus. The sera were obtained after 8 weeks on study. The circles indicate the neutralization titers of the individual sera of mice in each of the groups. The P value shown is the result obtained by comparing the geometric means of the two groups by Student t test.
FIG. 4.
FIG. 4.
Effect of gp160 cytoplasmic tail deletion on immunogenicity of VEE-HIV-1 env replicons. C3H/He mice were immunized with pGPm replicons expressing either gp140 or gp160ΔCT at 0, 2, 6, and 10 weeks, and sera were obtained at 12 weeks after the start of the study. Control sera were HNS2 (▪) and the negative reference serum (□).
FIG. 5.
FIG. 5.
Virus-neutralizing activity in sera of immunized mice is copurified with IgG. (Top) Sera from four immunized BALB/c mice from experiment II (Fig. 2) with comparatively high strain R2-neutralizing activity and from control mice in the same experiment (nonimmune IgG) were pooled, and IgG fractions were prepared as described in Materials and Methods. These materials were tested for neutralization of R2 envelope pseudotyped virus in comparison to the unfractionated immune serum pool. (Bottom) Similar immune globulin fractions were prepared from sera of C3H mice immunized with replicons expressing gp140 and tested for neutralization of SF162 envelope pseudotyped virus. The results shown in both panels are mean numbers of luminescence units obtained after virus incubation with the serum pool diluted 1:40 and IgG fractions of immune sera diluted to correspond to the concentrations in the serum pools from which they were derived. The nonimmune globulin preparations were tested at approximately the same IgG concentrations as the immune globulin preparations.
FIG. 6.
FIG. 6.
Comparative EIA binding and neutralizing activities of sera of mice of different strains immunized with VEE replicons expressing gp160 (closed circles) or gp140 (open circles) at 106 FFU/dose. Immunizations were given at 0, 1, and 2 weeks on study and approximately every 2 months thereafter. Blood was obtained for assay approximately 2 weeks after the third and each subsequent immunization. Neutralization assays were done by using pseudotyped viruses. Neutralization titers were determined as the highest serum dilutions that caused reductions in mean luminescence results of >50%.
FIG. 7.
FIG. 7.
Cross-reactivity of neutralizing activity in sera from gp160 immune C3H/He mice against primary HIV-1 envelope pseudotyped viruses. (A) The heights of the open columns indicate the geometric mean titers of six sera from immune mice against viruses pseudotyped with the R2 or SF162 envelope or one of four MACS donor envelopes. The circles indicate the 50% neutralization titers obtained with the individual sera. The closed columns indicate the titers obtained against the same viruses by using the HNS2 serum. In panel B, the results are shown for neutralization of viruses pseudotyped with the R2, Chinese clade C, and Chinese clade E (CRF-1) envelopes. The heights of the columns indicate the 50% neutralization titers of the immune mouse serum pool (□) and HNS2 (▪) and the geometric mean 50% neutralization titers of three sera from African donors infected with clade C viruses (▨) and three sera from Thai donors infected with clade E viruses (▧).
FIG. 8.
FIG. 8.
Neutralization of replication-competent clade B virus strain infections of cell cultures. Isolate IIIB infections were assayed in H9 cells, and strain BZ167 and US1 infections were assayed in PBMC. Amounts of p24 in culture supernatants were assayed 1 week postinfection. Results are expressed as percentages of p24 in medium from cells challenged with virus preadsorbed to nonimmune sera. Results are from three separate assays, each done in triplicate. Bars indicate mean percent neutralization in triplicate experiments.
FIG. 9.
FIG. 9.
Neutralizing activity in sera from New Zealand White rabbits immunized with VEE replicons expressing the HIV-1 strain R2 envelope. Rabbits were immunized by subcutaneous or intradermal inoculation of replicons expressing R2 gp140 (first four immunizations) or R2 gp160ΔCT (remaining immunizations) at weekly intervals for three inoculations and then at approximately 6-week intervals for four inoculations. The results shown are the 50% neutralization titers of each serum against viruses pseudotyped with SF162 (closed columns) or R2 (open columns). The tests were performed in triplicate, and the assay was repeated.
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References

    1. Beirnaert, E., P. Nyambi, B. Willems, L. Heyndrickx, R. Colebunders, W. Janssens, and G. van Der. 2000. Identification and characterization of sera from HIV-infected individuals with broad cross-neutralizing activity against group M (env clade A-H) and group O primary HIV-1 isolates. J. Med. Virol. 62:14-24. - PubMed
    1. Browning, J., J. W. Horner, M. Pettoello-Mantovani, C. Raker, S. Yurasov, R. A. DePinho, and H. Goldstein. 1997. Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc. Natl. Acad. Sci. USA 94:14637-14641. - PMC - PubMed
    1. Caley, I. J., M. R. Betts, N. L. Davis, R. Swanstrom, J. A. Frelinger, and R. E. Johnston. 1999. Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy. Vaccine 17:3124-3135. - PubMed
    1. Caley, I. J., M. R. Betts, D. M. Irlbeck, N. L. Davis, R. Swanstrom, J. A. Frelinger, and R. E. Johnston. 1997. Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector. J. Virol. 71:3031-3038. - PMC - PubMed
    1. Charles, P. C., K. W. Brown, N. L. Davis, M. K. Hart, and R. E. Johnston. 1997. Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate. Virology 228:153-160. - PubMed

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