John R. Mascola

Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies.

The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.

Human skin Langerhans cells are targets of dengue virus infection

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukoctye antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60–80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.

A phase I/II trial of HIV SF2 gp120/MF59 vaccine in seronegative Thais.

Abstract Fifty-two human immunodeficiency virus type 1, seronegative Thai adults from the community were enrolled in a double-blind, placebo controlled, phase I/II trial of HIV SF2 gp120/MF59 vaccine to determine the safety and immunogenicity of this recombinant, B clade, HIV envelope protein vaccine. Twenty-six subjects were enrolled at each of two sites in Thailand, Bangkok and Chiang Mai. Twelve subjects received placebo and 40 subjects received vaccine (50 μg). Subjects were immunized according to one of two schedules, 0, 1 and 4 or 0, 1 and 6 months. The frequency of adverse reactions was not different between placebo and vaccine subjects, nor between immunization schedules. Of vaccinees, all developed high-titer binding antibody to the immunogen (rgp120), 39 developed neutralizing antibody (NA) responses against homologous virus (HIV-1SF2), and 22 developed NA against heterologous virus (HIV-1MN). No subject demonstrated intercurrent HIV infection, however screening EIA reactivity occurred in 27% of recipients. Thus, this candidate HIV vaccine was found to be safe and immunogenic in Thai adults, laying the foundation for development of a subtype E construct in this population.

Cellular Immunity Elicited by Human Immunodeficiency Virus Type 1/ Simian Immunodeficiency Virus DNA Vaccination Does Not Augment the Sterile Protection Afforded by Passive Infusion of Neutralizing Antibodies

ABSTRACT High levels of infused anti-human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibodies (MAbs) can completely protect macaque monkeys against mucosal chimeric simian-human immunodeficiency virus (SHIV) infection. Antibody levels below the protective threshold do not prevent infection but can substantially reduce plasma viremia. To assess if HIV-1/SIV-specific cellular immunity could combine with antibodies to produce sterile protection, we studied the effect of a suboptimal infusion of anti-HIV-1 neutralizing antibodies in macaques with active cellular immunity induced by interleukin-2 (IL-2)-adjuvanted DNA immunization. Twenty female macaques were divided into four groups: (i) DNA immunization plus irrelevant antibody, (ii) DNA immunization plus infusion of neutralizing MAbs 2F5 and 2G12, (iii) sham DNA plus 2F5 and 2G12, and (iv) sham DNA plus irrelevant antibody. DNA-immunized monkeys developed CD4 and CD8 T-cell responses as measured by epitope-specific tetramer staining and by pooled peptide ELISPOT assays for gamma interferon-secreting cells. After vaginal challenge, DNA-immunized animals that received irrelevant antibody became SHIV infected but displayed lower plasma viremia than control animals. Complete protection against SHIV challenge occurred in three animals that received sham DNA plus MAbs 2F5 and 2G12 and in two animals that received the DNA vaccine plus MAbs 2F5 and 2G12. Thus, although DNA immunization produced robust HIV-specific T-cell responses, we were unable to demonstrate that these responses contributed to the sterile protection mediated by passive infusion of neutralizing antibodies. These data suggest that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection.

Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC)

The serum titration neutralization assay described in Chapter 33 utilizes a constant amount of infectious virus and indirectly estimates the antibody-mediated reduction in infectious virus by measuring p24 antigen expressed by human peripheral blood mononuclear cells (PBMC). The assay assumes a direct relationship between expressed p24 antigen and the number of target PBMC infected. Therefore, the optimal time of p24 antigen measurement may vary due to different growth kinetics among viruses. An infectivity reduction assay (IRA) directly measures the effect of antibody on virus endpoint infectious titer. The IRA is more labor-intensive and requires more serum than the antibody titration assay, and it is generally done with a single dilution of antibody. However, it has the important advantage of directly measuring the effect of antibody on virus median tissue culture infectious dose (TCID(50)). In addition, the IRA is unaffected by variation in virus growth kinetics and therefore is theoretically more suitable for comparing neutralization among diverse virus isolates. The assay described below calculates virus TCID(50) by serially diluting virus stock in quadruplicate wells with phytohemagglutinin (PHA)-stimulated PBMC. Wells are scored positive or negative for HIV-1 infection by measuring expressed p24 antigen after eight days in culture. Results are expressed as the antibody-mediated reduction in virus TCID(50) (i.e., ratio of virus TCID(50) in control sera to TCID(50) in test sera).

Determination of Syncytium-Inducing Phenotype of Primary HIV-1 Isolates Using MT-2 cells

HIV-1 is routinely isolated by cocultivation of patient PBMC with mitogen-stimulated HIV-uninfected donor PBMC (see Chapter 1 ). In this culture system, HIV-1 primarily replicates in CD4(+) T-lymphocytes, and such viruses are termed clinical or primary isolates. As early as 1986, the in vitro replicative capacity and cell tropism of primary HIV-1 isolates were shown to be important in the pathophysiology of HIV-1 infection (1). High replication capacity in PBMC and virus growth and syncytium formation in neoplastic T-cell lines were found to correlate with severity of HIV-1-disease (2-5). Compared to syncytium-inducing (SI) isolates, nonsyncytium-inducing (NSI) strains did not replicate in neoplastic T-cell lines and showed preferential replication in cells of the monocyte-macrophage lineage (6,7). Thus, NSI viruses have often been termed macrophage tropic, whereas SI strains are termed T-cell line tropic.

Determination of HIV-1 Chemokine Coreceptor Tropism Using Transduced Human Osteosarcoma (HOS) Cells

CD4 was identified in 1984 as the receptor for HIV-1 (1,2). However, it was soon apparent that a second receptor was necessary for HIV-1 infection of CD4(+) cells. This coreceptor was first identified by Berger and colleagues who showed that fusion and entry of T-cell line-adapted strains of HIV-1 into CD4(+) cells were mediated by a member of the seven transmembrane chemokine receptor family (3). This protein was initially termed fusin and later found to be the α-chemokine receptor CXCR4. Subsequent reports by several laboratories rapidly identified a β-chemokine receptor, CCR5, that mediated entry of macrophage tropic HIV-1 isolates into CD4(+) cells (4-8). Since these initial reports, our understanding of HIV-1 cell entry has continued to evolve. It now seems clear that primary HIV-1 strains with a nonsyncytium-inducing pheno-type (in MT2 cells) utilize the CCR5 coreceptor and are designated R5, whereas syncytium-inducing viruses preferentially utilize CXCR4, but may be dual tropic and are designated either X4 or X4R5, respectively (9,10). Some viruses also appear to be able to utilize chemokine receptors CCR2B and CCR3 (7-10). In addition, the role of chemokine coreceptors in the pathophysiology of HIV-1 infection and disease progression is just beginning to be understood. Individuals who are homozygous for a 32-bp deletion in the CCR5 gene are only rarely found to be HIV-1-infected, and the heterozygous CCR5/Δccr5 genotype has been associated with a survival advantage against HIV-1 disease progression (11-14).