Lymphoid activation: a confounding factor in AIDS vaccine development?

Jennifer Richardsonb,1, Sophie Broche1, Sandrine Baud1, Thierry Leste-Lasserre1, Françoise Féméniac,1, Daniel Levyd,1, Anne Moraillon1, Gianfranco Pancinoe,1 and Pierre Sonigo1

Génétique des Virus, Institut Cochin (INSERM U567 CNRS UMR 8104), 22 rue Méchain, 75014 Paris, France1
Immunopathologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique (INRA)2 and UMR INRA ENVA AFSSA 1161 de Virologie, Ecole Nationale Vétérinaire d’Alfort (ENVA)3, Maisons-Alfort, France

Author for correspondence: Pierre Sonigo. Fax +33 1 40 51 64 30. e-mail sonigo{at}cochin.inserm.fr


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In a previous vaccination trial, inoculation of env gene DNA failed to elicit a detectable antibody response, yet accelerated virus dissemination in most immunized cats following challenge with feline immunodeficiency virus. This result raised the possibility that cell-mediated immune responses had given rise to immune-mediated enhancement of infection. Since high-level replication of immunodeficiency viruses in lymphocytes requires cellular activation, antigen-specific responses or non-specific polyclonal activation may have increased the frequency of optimal target cells. In the present DNA vaccination trial, although designed so as to minimize non-specific polyclonal activation, immune-mediated enhancement was nonetheless observed in certain immunized cats. Moreover, rapid virus dissemination in vivo was associated with the presence of T-helper responses prior to challenge, and was linked to increased susceptibility of lymphocytes to ex vivo infection. Immune activation may thus be a confounding factor in vaccination against lentivirus infection, diminishing vaccine efficacy and giving rise to immune-mediated enhancement.


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In the effort to develop a vaccine against the human immunodeficiency viruses (HIV), it would be highly instructive to obtain vaccines that afford protection against the simian and feline immunodeficiency lentiviruses (SIV and FIV). Unfortunately, vaccines tested so far in animal models, with the exception of live attenuated vaccines, have only on rare occasions provided substantial protection against pathogenic immunodeficiency viruses (Hesselink et al., 1999 ; Johnston, 2000 ). Indeed, in several vaccination trials conducted in different laboratories, FIV disseminated more rapidly or established higher levels of viraemia in immunized cats than in unimmunized control animals after virulent challenge (Hosie et al., 1992 ; Huisman et al., 1998 ; Karlas et al., 1998 , 1999 ; Lombardi et al., 1994 ; Richardson et al., 1997 ; Siebelink et al., 1995 ). These results would appear to suggest that vaccination, while capable of inducing immune responses that contain virus replication, may also induce deleterious immune responses that enhance lentivirus dissemination.

Numerous studies have shown that certain antibodies directed against HIV type 1 (HIV-1) can enhance virus replication in various cell-culture models (Eaton et al., 1994 ; Homsy et al., 1989 ; Jouault et al., 1989 ; Kostrikis et al., 1996 ; Robinson et al., 1987 , 1990 , 1991 ; Schutten et al., 1995 ; Sullivan et al., 1995 , 1998 ; Takeda et al., 1988 ) or are associated with disease progression (Fust et al., 1994 ; Homsy et al., 1990 ; Montefiori et al., 1991 , 1996 ; Toth et al., 1991 ) or maternal transmission (Lallemant et al., 1994 ; Pancino et al., 1998 ). Nevertheless, not all instances of immune-mediated enhancement can be attributed to enhancing antibodies. In our laboratory, plasma virus RNA was detected substantially earlier in cats immunized with env gene DNA than in control cats (Richardson et al., 1997 ), though measurable levels of FIV-specific antibodies were not elicited prior to challenge, raising the possibility that the cell-mediated immune response and, in particular, concomitant lymphoid activation had accelerated the course of primary infection.

High-level replication of immunodeficiency viruses in T lymphocytes requires lymphoid activation (Ascher & Sheppard, 1990 ; Bukrinsky et al., 1991 ; Zack et al., 1990 ), and such activation may limit virus production during natural infection. Indeed, in the chronic stage of HIV-1 infection, exogenous factors that induce immune activation, such as contemporaneous infections (Bentwich et al., 2000 ; Bush et al., 1996 ; Goletti et al., 1996 ), immunization (Brichacek et al., 1996 ; O’Brien et al., 1995 ; Ortigao-de-Sampaio et al., 1998 ; Ostrowski et al., 1998 ; Stanley et al., 1996 ; Staprans et al., 1995 ) and administration of cytokines (Davey et al., 1997 ; Kovacs et al., 1995 ), create bursts of virus replication. Should the level of lymphoid activation also be limiting during the initial stages of natural infection, early virus dissemination could be accelerated under conditions where activation is enhanced.

In our previous DNA vaccination trial, cats were challenged shortly after a series of closely spaced DNA injections and thus presumably during the effector phase of the immune response, when the number of activated lymphocytes, both antigen-specific and non-specific, ought to have been substantially elevated. In the absence of potent antiviral effector functions, amplification of target cells may have given rise to the accelerated infection observed in immunized animals. In the present FIV vaccination trial, we have attempted to decrease the potentially deleterious effect of vaccine-related polyclonal activation by prolonging the intervals between DNA injections and between the final DNA injection and challenge. The vaccine vectors and vaccination schedule are represented schematically in Fig. 1. In the plasmid vector pCMV-env, expression of the env gene of the 34TF10 molecular clone of FIV (Talbott et al., 1989 ) was placed under the transcriptional control of the CMV-IE promoter, by PCR amplification of the sequence encoding both the surface (SU) and transmembrane glycoproteins and insertion in the PstI and BamHI sites of the VR1012 plasmid (Vical Inc.). In the vector pCMV-S (Davis et al., 1993 ), transcription of the S region of the hepatitis B virus (HBV) envelope gene was placed under the control of the CMV-IE promoter, permitting expression of a heterologous antigen, the S antigen of HBV (HBVsAg). Plasmid DNA was purified by using commercially available materials (Plasmid megakit, Qiagen) and endotoxin was removed by extraction with Triton X-114 (Aida & Pabst, 1990 ).



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Fig. 1. (a) Schematic diagram of pCMV-env and pCMV-S vectors respectively expressing FIV envelope glycoproteins and HBVsAg. In pCMV-env, the full-length env gene encoding surface (SU) and transmembrane (TM) glycoproteins is placed under the transcriptional control of the CMV IE promoter/enhancer (CMV P/E) and is flanked by the 5' untranslated region of CMV (CMV 5' UT) and a bovine growth hormone (BGH) sequence providing a polyadenylation signal. In pCMV-S, the S antigen of HBV is placed under the transcriptional control of the CMV P/E, with a polyadenylation signal furnished by the 3' region of HBV. (b) DNA vaccination schedule. Timing of DNA inoculations and virus challenge is indicated by single- and double-shafted arrows, respectively. The first two inoculations of pCMV-env (V1 {alpha}, V2 {alpha}) preceded those of pCMV-S (V1 {beta}, V2 {beta}).

 
Twenty 4-month-old specific-pathogen-free cats (IFFA-CREDO) were assigned randomly to two test groups (1 and 2) and two control groups (3 and 4) each containing four cats. Groups 1 and 2 were inoculated with pCMV-env by combined intradermal and intramuscular (i.d./i.m.) routes or targeted mesenteric lymph node (l.n.) inoculation, respectively. As controls, groups 4 and 3 were respectively inoculated i.d./i.m. with pCMV-S or with plasmid DNA (pUC18) devoid of a eukaryotic transcriptional unit. Groups 1, 3 and 4 received three injections (V1–V3) of 400 µg DNA at 0, 8 and 24 weeks (groups 1 and 3) or at 10, 19 and 24 weeks (group 4), as well as a single injection of 2 mg DNA (V4) 21 weeks after V3. In groups 1, 3 and 4, half of the DNA was injected i.m. at two sites and half i.d. at six sites. By contrast, in group 2, the DNA was injected in the mesenteric lymph node at a single site following surgical intervention. Group 2 received three injections (V1–V3) of 400 µg DNA at weeks 0, 8 and 24 and a single injection of 1 mg DNA (V4) 21 weeks after V3.

Similar to the previous DNA vaccination trial, humoral responses directed against envelope glycoproteins were virtually undetectable prior to challenge, while antibodies directed against the HBVsAg, assayed using commercially available reagents (MONOLISA Anti-HBs 3.0, Sanofi Diagnostics Pasteur), were detected in only one of four cats (Mexique) immunized with pCMV-S (data not shown). In an attempt to identify cell-mediated immune correlates of enhancement, T-helper (Th) responses directed against the SU glycoprotein were assayed in PBMC isolated 2 weeks after the fourth DNA injection.

For metabolic labelling with [3H]thymidine, PBMC were resuspended in RPMI 1640 medium containing 10% foetal calf serum, 10 mM HEPES, 2 mM glutamine, antibiotics and 50 µM {beta}-mercaptoethanol (complete RPMI) supplemented with 1% feline plasma. Cells (4x105 cells per well) were cultured in triplicate wells of transfer tubes (Costar) in the presence of immunoaffinity-purified FIV SU glycoprotein or medium alone, as well as in the presence of 10 µM concanavalin A, as a control for cell viability, in a final volume of 200 µl. Fifty µl volumes were removed after 24 and 48 h for assay of IL-2. After 72 h, the cells were pulsed with [3H]thymidine (1  µCi per well) and cells were collected onto glass filter paper with a semi-automated cell harvester (Scatron) after overnight incubation. Incorporation of [3H]thymidine was assessed by liquid scintillation counting.

For biological assay of IL-2, the IL-2-dependent CTL-L cell line was adjusted in complete RPMI to 2x105 cells per well and 50 µl volumes were added to the PBMC supernatants and cultivated for 16–24 h. The cells were then pulsed with [3H]thymidine as described above. Although proliferative responses were not discernible in any of the cats, antigenic stimulation gave rise to a substantial production of IL-2 in PBMC of two cats (Michelle and Mauritanie) inoculated l.n. with env DNA (Table 1).


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Table 1. The responses and susceptibility of PBMC to ex vivo infection

 
In order to determine whether lymphocytes of immunized cats exhibited enhanced susceptibility to FIV infection ex vivo, PBMC were isolated from vaccinated and control cats 2 weeks after the fourth DNA injection and exposed to FIV without mitogenic activation, either following incubation with SU glycoprotein or without any antigenic stimulation. In the absence of mitogenic and antigen-specific activation, the level of cellular activation was limiting for virus propagation, and the level of virus replication therefore provided an indirect measure of vaccine-related lymphoid activation. Antigen-specific stimulation, i.e. incubation with SU glycoprotein, was expected to induce immune activation and thereby enhance ex vivo infection in PBMC containing antigen-specific memory lymphocytes.

Feline PBMC were resuspended in complete RPMI containing 1% feline plasma. Cells (4x105 cells per well) were cultured in triplicate wells of transfer tubes in the presence of immunoaffinity-purified SU glycoprotein or medium alone, in a final volume of 200 µl. After 24 h, the PBMC were infected by addition of 25 TCID50 of the Wo strain of FIV (Moraillon et al., 1992 ). After a further 24 h, the virus inoculum was removed by washing the cells twice with 0·5 ml RPMI 1640 medium. The PBMC were then resuspended in 200 µl complete RPMI containing 5 µg/ml concanavalin A and 100 U/ml recombinant IL-2 and transferred to wells of 96-well microtitre plates. Half the medium was replaced 4 days after infection. At 4 and 7 days post-infection, aliquots of 10 µl were removed for assay of reverse transcriptase activity. Quantitative densitometry of autoradiography film was performed by using the program NIH Image version 1.54.

For cats immunized with env gene DNA, the extent of virus replication in the absence of antigen-specific stimulation was particularly high in PBMC isolated from two cats (Michelle and Mauritanie) of group 2 and, notably, the only two cats for which Th responses were detected (Table 1). Moreover, even higher levels of replication were obtained after incubation of PBMC from these two cats with SU glycoprotein. For cats of the two control groups, virus replication was particularly efficient in the PBMC of one cat (Mexique) of group 4, immunized with DNA encoding HBsAg, possibly because of immune activation elicited against this heterologous antigen.

Following homologous challenge by intraperitoneal injection of 10 median cat infectious doses (CID50) of the Petaluma strain (Pedersen et al., 1987 ) of FIV (stock provided by M. Hosie, University of Glasgow, UK), prepared from the supernatant of the chronically infected FL-4 cell line (Yamamoto et al., 1991 ), virus load in cats was assessed as plasma virus RNA by competitive RT–PCR (Richardson et al., 1998 ). Virus RNA was detected in the plasma of all but two cats, Moto and Mado, respectively from groups 1 and 3. Peak viraemia occurred between 32 and 36 days for control group 3 or between 25 and 46 days for both control group 4 and immunized group 1 (Table 2). No statistically significant differences were observed in plasma virus load upon pairwise comparison of these groups at any time point examined.


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Table 2. Development of plasma virus load

 
By contrast, virus dissemination was accelerated in the group immunized by l.n. inoculation of env gene DNA (group 2). Virus RNA was detectable in one cat (Michelle) as early as 7 days after challenge, and peak levels of viraemia were obtained earlier than in most control cats, at 18 (Mauritanie), 21 (Michelle) and 28 (Mathilde and Motte) days after challenge (Table 2). Virus load was higher than that of the control groups (3 and 4) throughout the early phase of infection. Moreover, when the level of virus RNA, determined as cumulative area under the curve (Dawson, 1994 ), was considered at 25 days post-infection, the virus load of group 2 was significantly greater than that of control group 3 (P=0·0304) and control groups 3 and 4 combined (P=0·0219).

In the present study, challenge was performed 10 weeks after the final DNA injection, so as to avoid the effector stage of the immune response. Under these conditions, and in contrast with the results of our previous trial, virus dissemination was not accelerated in cats vaccinated with env gene DNA by the i.d./i.m. routes. Immune-mediated enhancement was, however, observed in cats immunized by l.n. inoculation. Intriguingly, Th activity was restricted to the two cats of this immunization group with particularly rapid kinetics of infection. Moreover, rapid virus dissemination in vivo was linked to increased susceptibility of lymphocytes to ex vivo infection, as would be expected if cellular immunity underlies immune-mediated enhancement. While, given the small number of animals in question, far-reaching conclusions cannot be drawn, these results are consistent with the hypothesis, already formulated (Schwartz, 1994 ), that induction of a virus-specific Th response might provide a pool of highly susceptible target cells upon lentivirus infection and, paradoxically, enhance infection.

These results underscore an apparent paradox in vaccination against lentiviruses: namely, that effective vaccination requires lymphoid activation, but lentiviruses cannot replicate efficiently in lymphocytes unless they are activated. Immune activation may therefore be a confounding factor in vaccination against lentivirus infection, diminishing vaccine efficacy and giving rise to immune-mediated enhancement of infection in the absence of potent counteracting protective responses. At present, the consequences of such activation for susceptibility to infection and disease progression are unknown. A better understanding of immune enhancement of infection is therefore necessary not only to improve upon current vaccines, but also to appreciate the risk that this phenomenon represents in human AIDS trials.


   Acknowledgments
 
We thank Virbac Laboratories for continuous support and the staff of the cattery of the Ecole Nationale Vétérinaire for expert assistance. This work was supported by grants from the Agence Nationale de la Recherche sur le SIDA, the Ensemble Contre le SIDA/Sidaction and the European Economic Community (Biomed 2).


   Footnotes
 
b Present address: UMR 1161 INRA ENVA AFSSA de Virologie, ENVA, Maisons-Alfort, France.

c Present address: UMR 956 INRA ENVA AFSSA de Biologie Moléculaire et Immunologie Parasitaires et Fongiques, Maisons-Alfort, France.

d Present address: Génopole, Evry, France.

e Present address: Unité de Biologie des Rétrovirus, Institut Pasteur, Paris, France.


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Received 2 January 2002; accepted 4 June 2002.