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Conclusion:

These passive immunization data give proof of IgG-mediated enhanced virus acquisition after mucosal exposure – a potential concern for antibody-based AIDS vaccine development.

Discussion

Here we showed: i) enSHIVIG, when passively administered to macaques, enhanced virus acquisition and significantly lowered the amount of virus needed to achieve viremia compared to naive controls; ii) ex-vivo enSHIVIG testing in the presence of active complement revealed significant C’-ADE activity that was abrogated by C’ heat inactivation or anti-CD21 mAb. These results indicate that antibodies generated during early-stage HIV/SHIV infection may increase host susceptibility and facilitate virus acquisition and early dissemination.

Previously [21], we had treated macaques biweekly with different intravenous doses of SHIVIG, the polyclonal high-titer neutralizing IgG, in order to link in-vitro neutralization titers with prevention of mucosal SHIV acquisition. Unexpectedly, animals pretreated with low-dose SHIVIG (25 mg/kg) had more viral quasispecies compared to untreated controls – implying increased SHIV transmission. Despite good SHIVIG neutralizing activity in TZM-bl cells, enhancement was observed in the presence of active complement in CR2/CD21-expressing SupT1.R5 cells that was abrogated by complement heat inactivation [21]. Together, these findings reinforce our current data that weakly or non-neutralizing neutralizing IgG may enhance mucosal SHIV acquisition through mechanisms dependent on complement activation.

It is intriguing to compare the 3.4-fold enhanced mucosal SHIV-1157ipd3N4 acquisition we report here with the magnitude of in-vitro HIV enhancement by Willey et al.[18] who measured C’-ADE in CR2-expressing SupT1/R5 cells using paired autologous early-stage sera/HIV isolates. Enhancement ranged from 8- to 236-fold and was lower when assessed with heterologous virus isolates. Differences in the order-of-magnitude of HIV C’-ADE reported [18] and our 3.4-fold lowering of the SHIV challenge dose needed to persistently infect enSHIVIG-pretreated macaques can be ascribed to CR2 expression by all SupT1.R5 cells used for in-vitro assays. In vivo, however, CR2 is expressed only by select cell populations, such as B cells, follicular dendritic cells, and according to a recent report [33], on naive CD4+ and CD8+ T cells.

In addition to C’-ADE, in-vitro assays have revealed another mechanism: Fc receptor-mediated ADE (FcR-ADE) [11,13,34–37] (reviewed in [38,39]). Monocyte/macrophage-derived cell lines expressing different FcRs were used to demonstrate FcR-ADE. Forthal et al.[40] provided indirect evidence of FcR-ADE from a Phase III AIDS vaccine trial; by subgroup analysis, a statistically significant association was noted between increased HIV acquisition and the Fc?RIIIa allele in vaccinees given monomeric gp120.

Our present data as well as those summarized above from prior studies have one common denominator: the IgGs were polyclonal. As such, we cannot distinguish between two possibilities for ADE: i) polyclonal IgG consists of a mixture inherently neutralizing and inherently enhancing antibodies; and ii) a given IgG neutralizes in one situation and enhances in another. This key issue can only be addressed by using mAbs – done in a seminal study by Kliks et al.[41] who examined the interaction of two different human anti-V3 mAbs with three different HIV-1 strains. Depending on the virus tested, the results yielded either neutralization, enhancement, or neither. Thus, well characterized mAbs are unpredictable in their interactions with different HIV strains. Enhancing antibodies have also been implicated in mother-to-child transmission of HIV in a number of studies [42–44]; some reports raised the possibility that enhancement may be linked to antibodies targeting HIV-1 gp41 [43–45].

Although different investigators have shown HIV ADE in various cell line-based assays over the years, whether such in-vitro data would translate into Antibody-Dependent Enhanced Virus Acquisition – ADE-VA – remained unsolved. Passive immunization of macaques with early-stage anti-SHIV IgG followed by intrarectal SHIV challenge gave proof-of-principle for increased virus acquisition and host susceptibility. AIDS vaccine development should consider the potential of ADE-VA due to vaccine-induced antibodies during experimental vaccine trials. To rule out this possibility, passive immunization with vaccine-induced antibodies could be used as a tool in biologically relevant animal models, that is, models that reflect key aspects of HIV transmission among humans, including i) tier 2 R5 challenge viruses carrying HIV-1 Env, ii) a nonhuman primate species, and iii) antibodies that are heterologous to the challenge viruses. The latter point is important since matched homologous virus/antibody systems will exaggerate neutralization and thereby mask potential enhancement by weakly or non-neutralizing antibodies. In the realistic setting of human vaccinees’ exposure to circulating HIV strains, an exact match between immunogen composition and the myriad of HIV quasispecies can never be expected.

Indirect evidence that vaccine-induced antibodies can have adverse effects comes from a feline immunodeficiency virus (FIV) study, where cats were vaccinated with various recombinant envelope glycoproteins [46]. Although neutralization in cell-line based assays was observed in plasma samples from some vaccinated groups, no virus-neutralizing antibodies were detected in the feline lymphocyte assay. Upon FIV challenge, cell-associated FIV loads were increased in the groups vaccinated with recombinant FIV Env glycoproteins compared to other groups or controls. Passive transfer of unfractionated plasma from groups with increased cell-associated FIV enhanced viral infection parameters in the recipients. While these data imply ADE, an influence of other factor(s) present in unfractionated plasma cannot be ruled out.

In sum, AIDS virus C’-ADE is real – as our passive immunization showed significant lowering of the virus dose needed to achieve viremia indicative of ADE-VA. As such, the current study with early-stage enSHIVIG confirmed our unexpected finding with late-stage SHIVIG, selected for maximal in-vitro tier 2 SHIV cross-neutralization, where low-dose pretreatment yielded sub-neutralizing anti-HIV Env IgG levels that significantly increased the number of transmitted viral quasispecies. Together, our data imply that decreasing anti-HIV Env neutralizing antibody titers could bring vaccinated individuals into a situation where ADE-VA prevails.

ADE-VA may be of concern for other pathogens, especially rapidly mutating RNA viruses susceptible to neutralization escape. Vaccine development will need to consider potential enhancement of host susceptibility to infection due to ADE [47,48]. We propose that our strategy – passive immunization with purified polyclonal IgG isolated from previously infected/vaccinated individuals, combined with in-vivo end-point virus titration to assess the amount of virus needed to achieve infection of naïve versus passively immunized animals, can play an important role in assessing the potential for ADE-VA.

https://www.uab.edu/medicine/gastroenterology/research/mhic/investigators/23-centers/mhic/76-christina-ochsenbauer-jambor-phd

Christina Ochsenbauer-Jambor, Ph.D.

Dr. Ochsenbauer-Jambor completed her undergraduate studies in biology at the Johann-Wolfgang-Goethe Universität, Frankfurt am Main in Germany in 1988. She then moved to the Ruprecht-Karls-Universität in Heidelberg, Germany, where she studied molecular biology (virology) and zoology (primate ethology) and earned her ‘Diplom’ (Master) of Biology (magna cum laude) in 1992. Her Master’s thesis focused on functions of the HIV-1 Env protein, with the work performed at the German Cancer Research Center (DKFZ) in Heidelberg. After completing her Ph.D. thesis entitled “Investigations concerning the function of the HIV-1 Vif protein: Generation of a cell-culture model system by using selectable, replication-competent HIV-1” at the DKFZ, she received her Ph.D. (magna cum laude) in virology and molecular biology from Ruprecht-Karls-Universität in 1996. During her undergraduate and graduate work, Dr. Ochsenbauer-Jambor was a recipient of two scholarships from the German National Merit Fundation. After postdoctoral fellowship (1996-2001) in retroviral glycoprotein intracellular trafficking in the Department of Microbiology at UAB, she joined the UAB faculty in 2002 as a Research Instructor in the same department. In 2003, she relocated to the Department of Medicine, Division of Hematology/ Oncology.

Dr. Ochsenbauer-Jambor investigates the role of DC-SIGN in the capture and trans-infection of HIV-1 with special emphasis on virion internalization pathways and the acquisition of selective DC-SIGN-dependent resistance to neutralizing antibodies.