| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden
2 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden
3 Mucosal Immunobiology & Vaccine Center (MIVAC), Department of Microbiology & Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
4 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence
Nils Y. Lycke
nils.lycke{at}microbio.gu.se
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Published online ahead of print on 11 September 2008 as DOI 10.1099/vir.0.2008/005470-0
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Mestecky et al., 2005). Vaccine-induced neutralizing antibodies (NAbs) that interfere with viral entry have been found to be the protective correlates of many other prophylactic antiviral vaccines (Parren et al., 2001; Wicker et al., 2007; Zignol et al., 2004). An HIV-1 vaccine that prevents or limits virus replication in the mucosa should reduce the risk of systemic viral dissemination (Haase, 2005; Maher et al., 2005; Mazanec et al., 1992) and may reduce the risk of human-to-human transmission (Kozlowski & Neutra, 2003; Mascola, 2003; Neutra & Kozlowski, 2006). However, the development of mucosal vaccines has been slow, owing mainly to the lack of safe and effective mucosal adjuvants. Furthermore, there is insufficient knowledge as to the best route for immunization of the genital tract: i.n., local vaginal, rectal or parenteral. Also, combinations of regimens, such as mucosal priming followed by systemic boost, or, systemic priming followed by mucosal boost, have not been exhaustively investigated (Vajdy et al., 2004). Finally, an overriding problem to the development of a protective vaccine against HIV-1 is the design of an envelope glycoprotein (Env) immunogen that is capable of inducing broadly NAbs (Burton et al., 2004, 2005; Karlsson Hedestam et al., 2008).
The HIV-1 Env glycoproteins consist of trimers of non-covalently associated gp120–gp41 heterodimers, critical for viral entry, and the sole targets for NAbs. Over the past decade, it has become apparent that unmodified monomeric Env variants (gp120) fail to elicit NAbs (Albrecht, 2003; Pitisuttithum et al., 2006). Therefore, in attempts to better mimic the native viral spike, soluble gp140 trimers containing full-length gp120 covalently linked to the gp41 ectodomain were designed (Binley et al., 2000; Kim et al., 2005; Sanders et al., 2002; Srivastava et al., 2003; Yang et al., 2000, 2002). Subsequent immunogenicity studies suggested that there was an incremental advance in elicitation of NAbs using soluble trimers as opposed to monomeric gp120 (Li et al., 2006; Yang et al., 2001). Other oligomeric Env gp140 proteins, with or without a deletion of the second major variable region of gp120 (
V2), have also been evaluated for their capacity to stimulate NAbs (Barnett et al., 2001; Derby et al., 2006; Xu et al., 2006a, b). Barnett et al. (2008) recently demonstrated that following combined mucosal and parenteral immunizations in non-human primates a gp140 molecule, lacking the V2 region, mediated protection against mucosal challenge with the homologous simian-human immunodeficiency virus (SHIV).
Mucosal delivery of vaccines in general has been hampered by the lack of effective and non-toxic adjuvants that function at mucosal sites and which promote both strong local mucosal IgA and serum IgG antigen-specific antibodies. Apart from the bacterial holotoxins, cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), few substances have proven effective mucosal adjuvants in experimental animal models (Lavelle et al., 2004; Levine & Sztein, 2004; Lycke, 2007). These molecules are AB5-complexes with an ADP-ribosylating A1-enzyme linked to a pentamer of B-subunits, which bind to the GM1-ganglioside, present on all nucleated cells (Hol et al., 1995). Because of the toxicity of these molecules and the reported side effects after i.n. administration, most notably Bell's palsy, it is unlikely that these holotoxins would be deemed safe enough for clinical use (Mutsch et al., 2004).
In fact, all holotoxins and their mutants with binding to the ganglioside receptors, carry a similar risk of negatively affecting the central nervous system following i.n. administration. Therefore, we have developed a non-toxic adjuvant based on the CTA1-enzymic activity, but without the promiscuous cholera B-subunit (CTB) binding to the ganglioside receptors (Agren et al., 1997). The CTA1-DD molecule has been extensively evaluated for its mucosal adjuvant activity in mice (Agren et al., 1997; Choi et al., 2002; De Filette et al., 2006; Eriksson et al., 2004). Importantly, we have previously demonstrated that i.n. administration of the CTA1-DD adjuvant does not affect nervous tissues and efficiently stimulates both systemic and mucosal antibody, and CD4+ and CD8+ T-cell responses to levels comparable to those seen with intact CT holotoxin (Eriksson et al., 2004; Simmons et al., 1999).
To date, the CTA1-DD adjuvant has proven highly effective for experimental i.n. immunizations against several infectious disease agents, including those from rotavirus, influenza virus, Helicobacter pylori and Mycobacterium tuberculosis (Akhiani et al., 2006; Andersen et al., 2007; Eliasson et al., 2008; McNeal et al., 2007). In the present study, we asked if CTA1-DD would augment HIV-1 Env-specific immune responses in both mice and non-human primates and whether antibody responses at mucosal sites were elicited. We also asked if the adjuvant effect of CTA1-DD was dependent on the MyD88 signalling pathway for toll-like receptor (TLR)-stimulation and we compared the adjuvant effect of CTA1-DD with those of three other adjuvants: Ribi, LTK63 and CpG oligodeoxynucleotides (ODN).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were bred at the Experimental Biology (EBM) unit at the University of Göteborg, Sweden. All mice were sex and age matched and kept under specific-pathogen-free conditions at the EBM or at the animal facility at the Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet, Sweden. Seven female cynomolgus macaques (Macaca fascicularis) of Chinese origin, 5–6 years old, were housed in the Astrid Fagraeus laboratory at the Swedish Institute for Infectious Disease Control, Stockholm, Sweden. Housing and care procedures were in compliance with the provisions and general guidelines of the Swedish Animal Welfare Agency and all procedures were approved by the Local Ethical Committee on Animal Experiments. The animals were housed in pairs in 4 m3 approved cages. They were habituated to the housing conditions for more than 6 weeks before the start of the experiment, and subjected to positive reinforcement training in order to reduce the stress associated with experimental procedures.
Expression and purification of Env immunogens.
The gp140-F trimers and gp120 monomers were produced as described previously (Grundner et al., 2004; Yang et al., 2002). Briefly, transient transfection of gp140-F plasmids into adherent 293F cells (Invitrogen), cultured in Dulbecco's modified Eagles medium (DMEM)/10 % heat-inactivated fetal bovine serum/0.1 mM MEM non-essential amino acid solution (Invitrogen) medium, was achieved using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Every other day serum-free 293 SFM II medium (Invitrogen) was replaced in the cultures as supernatants were collected daily until day 6 post-transfection. A three-step procedure was used to purify the His6-tag-containing trimers, including lentil-lectin affinity chromatography (GE Healthcare), eluted with PBS/1 M 
-β mannopyranoside/0.5 M NaCl/10 mM imidazole, and captured via the His-tag by nickel-chelation chromatography (GE Healthcare) and eluted with a 300 mM imidazole-containing PBS buffer, followed by separation of lower molecular mass proteins by gel filtration chromatography. The gp120 monomers were produced in Freestyle 293F suspension cells by using 293Fectin transfection reagent in Opti-MEM according to the manufacturer's instructions (Invitrogen). Supernatants from 4 days were centrifuged at 3500 g to remove cells or cell debris, filtered through a 0.22 µm filter and supplemented with Complete, EDTA-free protease inhibitor cocktail (Roche) and penicillin–streptomycin (Invitrogen) for storage at 4 °C until further purification. The gp120 monomers were purified by capturing the proteins in an IgG17b affinity column. All proteins were spun concentrated with Amicon Ultra 30 000 MWCO centrifugal Filter Devices (Millipore) to a concentration between 1 and 3 mg ml–1.
Immunizations.
Mice were immunized i.n. or intraperitoneally (i.p.) using different regimens of adjuvants and monomeric or trimeric HIV-1 Env. In brief, immunizations were performed using 10 µg Env together with 5 µg CTA1-DD with 10 day intervals between three, four or five i.n. inoculations or admixed with Ribi (MPL+TDM+CWS Adjuvant System, M6661; Sigma-Aldrich) for three i.p. injections. Alternatively, two immunizations with Env and CTA1-DD i.n. were followed by a boost with Env and Ribi i.p. When indicated mice were given 5 µg per dose of LTK63 (kind gift from Novartis) or CpG ODN (CyberGene AB) with 10 µg Env i.n.; control animals were immunized three times with PBS or three, four or five times with CTA1-DD alone or Env alone. Immunizations (i.n.) were given in 10 µl PBS in each nostril and i.p. injections in 100 µl PBS or 0.9 % saline (for Ribi). Serum was taken 10 days after each immunization, and spleens, bronchial alveolar lavage (BAL) and vaginal secretions were taken 10 days after the last immunization when sacrificing the mice. Monkeys were inoculated three times intramuscularly (i.m.) with 200 µg Env and Ribi in a total volume of 1 ml, divided between the left and right hind leg or i.n. with 200 µg Env and 50 µg CTA1-DD in PBS in a total volume of 100 µl divided between the nostrils. Blood samples were taken before and 2 weeks after each immunization. All immunizations and blood sampling were performed under sedation with ketamine 10 mg kg–1 i.m. (Ketaminol 100 mg ml–1; Intervet). The macaques were weighed and examined for swelling of lymph nodes and spleen at each immunization or sampling occasion.
Antibody determinations.
Determinations of antibody levels in mouse serum, BAL and vaginal secretions were done by ELISA as described previously (Eliasson et al., 2008; Forsell et al., 2007; Marks et al., 2007). Briefly, ELISA plates (Nunc) were coated with 1 µg insect cell (S2)-produced YU2gp120 ml–1 in 100 µl PBS at 4 °C overnight. The plates were blocked by incubation with PBS+2 % non-fat dried milk and 5 % fetal calf serum (FCS) (Sigma-Aldrich) at room temperature and then washed three times in PBS+0.2 % Tween 20 (Sigma-Aldrich). Serum was added in serial dilutions and incubated at room temperature before washing and incubating with goat anti-mouse total IgG-horseradish peroxidase (HRP) or IgG subclass-specific (Southern Biotech) antibody at dilution 1/5000 or goat anti-mouse IgA-HRP (Southern Biotech) at 1/500 dilution. After washing, the plates were developed using the OPD Fast kit (Sigma-Aldrich) and the optical density read at 450 nm using a Multiscan EX (MTX Lab systems). For determination of specific anti-Env responses in monkeys, Maxisorp microtitre plates (Nunc) were coated with 1 µg S2-produced gp120 ml–1 in 50 mM carbonate buffer (pH 9.6), overnight at 4 °C. The serum samples were serially diluted in PBS with 20 % FCS and 0.5 % Tween 20, and added to the plates after blocking with 20 % FCS in PBS. For measurement of IgA responses, the sera were first depleted of IgG using Protein A–Sepharose CL-4B (Amersham Pharmacia Biotech). After overnight incubation at 4 °C, bound IgG or IgA was detected with HRP-conjugated anti-human IgG (P0214; DakoCytomation) or anti-monkey IgA [GAMon/IgA(Fc)/PO; Nordic Immunological Laboratories], respectively, followed by the addition of O-phenylenediamine with H2O2 (DakoCytomation). The plates were washed six times with PBS supplemented with 0.5 M NaCl and 0.1 % Tween 20 between all steps. Substrate reactions were stopped with 2 M H2SO4 and optical density read at 492 and 620 nm. In mice, log10 titres were calculated as described previously (Eliasson et al., 2008) and in monkeys geometric means of end-point titres, defined as the last serum dilution giving an optical density value 2 SD above background optical density for pre-immune sera, was calculated.
Cellular responses.
One week after the last immunization, spleens were removed and single-cell suspensions were prepared and resuspended at 2x106 cells ml–1 in Hanks' buffered saline solution (Gibco). The cells were cultured in triplicates in 96-well microtitre plates (Nunc) in Iscove's medium (Biochrom KG), supplemented with 10 % heat-inactivated FCS (Biochrom), 50 µM 2-mercaptoethanol (Sigma-Aldrich), 1 mM L-glutamine (Biochrom) and 50 µg gentamicin (Sigma-Aldrich) ml–1 and cultured for 72 h at 37 °C in 5 % CO2 either alone or with 1 µM gp120 Env protein (KJ Ross-Petersen AB) or 10 µg tetanus toxoid (TT; Statens Serum Institute) ml–1. Specific cell proliferation to recall antigen or TT was assessed after 72 h of culture, by the addition of 1 µCi (37 kBq) per well [3H]thymidine (Amersham International) for the last 6 h of culture. The [3H]thymidine uptake was determined by using a scintillation counter (Beckman). Data were expressed as mean c.p.m±SEM of five mice per group in each experiment. Cytokine containing supernatants were collected after 96 h of cell culturing and stored at –70 °C until analysed. Briefly, gamma interferon (IFN-
), interleukin (IL)-4, IL-5 and IL-10 levels in supernatants were assessed by ELISA. Microtitre plates (Dynatech Laboratories) were incubated with 2.5 µg rat anti-mouse IFN- (BD Biosciences) ml–1 or 1–5 µg anti-mouse IL-4 (Endogen), IL-5 or IL-10 (R&D System) ml–1. The sample supernatants or recombinant mouse IFN-, IL-4, IL-5 or IL-10 (R&D System) standards were then added to the appropriate wells. Bound cytokines were detected by sequential incubations with a polyclonal rabbit anti-IFN- antiserum, or biotinylated mAb to mouse IL-4 (Endogen), IL-5 or IL-10 (R&D System) as described previously (Akhiani et al., 2002). The sensitivity of detection for the respective cytokine was 10 pg IFN- ml–1 and 5 pg IL-4, IL-5 or IL-10 ml–1.
Statistical analysis.
Wilcoxon rank sum test was used for independent samples for analysis of significance in all experimental groups, except for the IFN- values, which were compared by the one-tailed Student's t-test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Control mice were immunized i.n. with CTA1-DD alone or with Env alone. We also included a group of mice immunized twice with Env and CTA1-DD i.n. and then boosted once with Env and Ribi i.p. (Fig. 1a). We detected significant amounts of serum anti-Env IgG log10 titres in the mice immunized i.n. with Env and CTA1-DD while, in contrast, inoculation of CTA1-DD alone or Env alone did not induce any detectable anti-Env antibodies. Moreover, an i.p. boost of Env and Ribi in mice previously primed i.n. with CTA1-DD and Env effectively increased serum anti-Env titres, achieving 10-fold higher serum IgG titres than after three i.n. immunizations. This response was comparable to that obtained in mice immunized three times with Env and CTA1-DD i.p. or three times with Env and Ribi i.p. Strikingly, however, whereas CTA1-DD and Ribi were equally effective as parenteral adjuvants, only CTA1-DD given i.n. stimulated Env-specific mucosal IgA responses in the BAL (Fig. 1b). Thus, CTA1-DD was an effective mucosal adjuvant for stimulating HIV-1 anti-Env serum IgG and mucosal IgA antibodies following i.n. immunizations. Of note, since we found that irrespective of whether gp140 trimers or monomeric gp120 were used for immunizations similar results were obtained and, hence, monomeric gp120 was used in all subsequent experiments in mice.
|
|
). Both serum IgG and BAL IgG responses were substantially enhanced by the fourth i.n. immunization with Env and CTA1-DD and serum IgG levels were further boosted by the fifth immunization (Fig. 2a, b). The serum IgG antibody titres were markedly higher than those observed after three immunizations and comparable to those obtained with Ribi adjuvant given three times i.p. (Figs 1
and 2). There were also detectable anti-Env IgG responses in the vaginal secretions of some animals after the fourth and the fifth immunization (Fig. 2c, f). Most importantly, specific mucosal anti-Env IgA titres in BAL were substantially augmented by the fourth and fifth immunization with Env and CTA1-DD (Fig. 2e). There were no detectable Env-specific antibodies in serum, BAL or vaginal secretions after five inoculations of Env alone or CTA1-DD alone. Collectively, our data show that five i.n. immunizations with CTA1-DD adjuvant promoted the strongest HIV-1 anti-Env IgG and IgA antibody titres in both serum and mucosal secretions.
|
). Therefore, we analysed IgG1, IgG2a, IgG2b and IgG3 anti-Env responses in serum from i.n. (CTA1-DD and Env) and i.p. (Ribi and Env) immunized mice. We found that IgG1 serum antibodies dominated in both CTA1-DD and Ribi immunized mice (Fig. 3a). After three i.n. immunizations with Env and CTA1-DD only IgG1 anti-Env serum antibodies were detected. However, subsequent immunizations resulted in detectable IgG2a, IgG2b and IgG3 anti-Env titres (Fig. 3). Taken together both Th1- (IgG2a) and Th2-regulated (IgG1) IgG-subclasses were represented. Also, when comparing the i.n. and i.p. routes for the distribution of Env-specific IgG isotypes similar results were observed, indicating a balanced Th1 and Th2 activity in both the CTA1-DD and Ribi adjuvant systems.
|
) and cytokine responses (Fig. 4b) in mice immunized three times i.n. with Env and CTA1-DD adjuvant or with Env alone. Splenocytes were prepared 8 days after the third immunization and cultured together with recall antigen in vitro. Cell proliferation in response to Env protein was significantly enhanced in mice immunized with Env and CTA1-DD adjuvant, while non-adjuvant Env-immunized mice failed to respond (Fig. 4). The cytokine response as measured in the culture supernatants was dominated by IFN- (Fig. 4), while IL-4 and IL-5 were not detected (data not shown). Weak IL-10 responses were also found in Env plus CTA1-DD immunized mice (data not shown). Cells cultured with an unrelated antigen, TT, did not show proliferation or IFN--production above medium background, indicating that the response to HIV-1 Env was antigen-specific (Fig. 4a, b). These data demonstrated that the CTA1-DD adjuvant also significantly enhanced priming of HIV-1 Env-specific T-cell responses following i.n. immunizations.
|
; Guy, 2007; Kwissa et al., 2007; Pasare & Medzhitov, 2005). We asked if the adjuvant effect of CTA1-DD was retained in mice lacking MyD88, an adaptor protein for signalling used by multiple TLRs (Barton & Medzhitov, 2002; McGettrick & O'Neill, 2007; Underhill & Ozinsky, 2002). Wild-type (WT) C57BL/6 or MyD88–/– mice were immunized with Env and CTA1-DD i.n. For comparison, mice were given i.n. immunizations with CpG ODN, an adjuvant known to depend on TLR9 and MyD88 signalling, and LTK63, a detoxified mutant of the LT holotoxin with well documented mucosal adjuvant functions (Tritto et al., 2007). Following three i.n. immunizations, we found that all three adjuvants were equally effective in stimulating Env-specific serum IgG antibodies in WT mice (Fig. 5). However, as predicted, the CpG ODN adjuvant was completely ineffective in MyD88-deficient mice, whereas both CTA1-DD and LTK63 were potent adjuvants also in MyD88–/– mice (Fig. 5). Moreover, extended experiments in TLR4-deficient mice (signalling through the MyD88 and Trif-pathways) corroborated that CTA1-DD appears to exert its adjuvant effect in a TLR-independent fashion (N. Lycke, unpublished observation).
|
) as well as IgA responses (Fig. 6b, d) following two i.n. inoculations. These responses were boosted by a third immunization, either with Env and CTA1-DD i.n. or with Env and Ribi i.m. Although, the monkeys inoculated three times i.m. with Env and Ribi exhibited stronger antibody responses, we conclude that i.n. immunizations with Env and CTA1-DD effectively primed Env-specific IgG and IgA antibody responses. Importantly, no side effects were observed in monkeys that received i.n. CTA1-DD. Thus, these first preliminary results in non-human primates support the fact that the CTA1-DD adjuvant is a safe and effective mucosal vaccine adjuvant.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The CTA1-DD adjuvant was previously shown to be non-toxic. It does not drive inflammation at the site of application and, importantly, it does not bind GM1-ganglioside receptors or accumulate in the central nervous system following i.n. administration (Eriksson et al., 2004). The latter effect was, in fact, the reason for the recent withdrawal from the market of an LT-adjuvant i.n. influenza vaccine as vaccinees displayed an increased incidence of Bell's palsy (Mutsch et al., 2004). Because CTA1-DD lacks the CTB domain, it cannot bind to the GM1-ganglioside receptors (Agren et al., 1997). Such a risk, though, is highly likely also with mutant derivatives of CT or LT, such as i.n. vaccines adjuvanted by LTR192R or the LTK63 mutant (Dickinson & Clements, 1995; Stephenson et al., 2006). Here, we compared side by side the adjuvant potency of CTA1-DD with that of LTK63 and found that the augmenting effects on specific serum IgG-responses were comparable. A similar result has previously been reported when comparing CTA1-DD with LTR192G for mucosal immunizations with rotavirus VP6 (McNeal et al., 2007). Given, the constraints of using GM1-binding holotoxins and their mutant derivatives as vaccine adjuvants in the clinic we propose that CTA1-DD may replace these mucosal adjuvants. This may rekindle interest in i.n. administration as a primary route for safe and effective mucosal immunization.
The relative roles of serum and mucosal IgG and IgA for providing protective responses against vaginal or rectal transmission of HIV-1 remains poorly understood. While recent years have provided a better understanding of the early events and different cell types employed by the HIV-1 virus to infect the genital tract (reviewed by Hladik & McElrath, 2008), questions still remain as to what is the role of strong vaccine-induced mucosal immunity for protection (Haynes & Shattock, 2008). A study by Barnett et al. (2008) showed that mucosal HIV-1 Env protein immunizations can stimulate protective immune responses against a homologous vaginal SHIV challenge. These data provided proof of principle and suggested that mucosal priming followed by parenteral booster immunization may be a promising regimen to stimulate protective immunity against HIV-1. The results presented in the current study showed that mucosal administration of HIV-1 Env and CTA1-DD adjuvant effectively primed anti-Env B-cell responses in both mice and in cynomolgus monkeys. Previous studies have reported on potentially important protective roles of specific mucosal IgG and IgA against HIV-1 (Bergmeier & Lehner, 2006; Mazzoli et al., 1997, Neutra & Kozlowski, 2006; Vajdy et al., 2004; Veazey et al., 2003). However, definitive evidence that Env-specific IgA exerts a protective role still awaits to be shown. Indeed, the need for better and more standardized methods to measure Env-specific antibodies in mucosal secretions was highlighted in a recent workshop on mucosal immunity and HIV/AIDS vaccines (Girard et al., 2008). There is a general agreement that the addition of better quantitative and qualitative methods for the detection of specific antibody-secreting cells in mucosal biopsies is much warranted (Montefiori et al., 2007; Shattock et al., 2008).
The CTA1-DD was recently shown to greatly augment protective immune responses against influenza A virus in a mouse model using the conserved M2e-epitope as the vaccine immunogen (Eliasson et al., 2008). This model largely reflects the protective efficacy of anti-M2e antibodies and, thus, indicates that CTA1-DD may be particularly suitable for further exploration as an adjuvant for modulating humoral immune responses (Lycke, 2007). Most adjuvants are derived from bacteria or viruses and are recognized by pattern recognition receptors (Barton & Medzhitov, 2002; Harris et al., 2006; McGettrick & O'Neill, 2007; Underhill & Ozinsky, 2002). In particular, the TLR-family of receptors are considered key elements in recognition of lipopolysaccharide (LPS), muramyldipeptide, flagellin, CpG and many other molecules with adjuvant function (Harris et al., 2006). However, the bacterial holotoxins use ganglioside receptors to target cells and when administered as adjuvants they will bind also to antigen-presenting cells via these receptors (Grdic et al., 2005). Whether the holotoxins engage the TLR family of receptors for their adjuvant function in vivo has not been investigated previously. However, one study reported that the cytokine-inducing effect in macrophages–monocytes of a B-subunit of an E. coli type LT-IIb was TLR2-dependent, while this receptor was not required for cytokine production by the LT-IIb holotoxin (Liang et al., 2007). Thus, a possible role of gangliosides as co-receptors to TLR2 signalling was discussed by these authors, though the data does not support a TLR-involvement in the adjuvant effect of the LT-IIb holotoxin. We found that neither CTA1-DD nor LTK63 were dependent on the MyD88 adaptor protein and subsequently did not use this pathway for exerting adjuvant effects in vivo. The MyD88–/– mice demonstrated comparable serum anti-Env IgG titres as seen in WT mice. By contrast, only WT, but not MyD88–/– mice, given the CpG ODN adjuvant developed specific anti-HIV-1 Env serum antibodies, consistent with previous findings that this adjuvant is dependent on TLR9 (Hemmi et al., 2003). Moreover, the adjuvant effects of CTA1-DD and of intact CT holotoxin were also unablated in TLR4–/– mice as well as in double TLR4/MyD88-deficient mice (N. Lycke, unpublished observation). However, Ribi contains monophospholipid (MPL), a synthetic analogue of LPS that activates target cells via TLR4 and signals via both the MyD88 and Trif pathways (Baldridge et al., 2004; Vogel, 1995). As it was recently reported that the adjuvant effect of Ribi was intact in mice lacking both MyD88 and Trif, it follows that the adjuvant effect can be independent of TLR signalling, even though MPL is strictly TLR4-dependent in its other biological functions (Gavin et al., 2006). Therefore, in future experiments we will also address the TLR-independence of CTA1-DD in mice double deficient for MyD88 and Trif (Beutler, 2007).
This is the first evaluation of the adjuvant effect of CTA1-DD in non-human primates. Although, the study is quite preliminary, it indicates that CTA1-DD exerts adjuvant effects in non-human primates. Monkeys immunized two or three times with Env and CTA1-DD i.n. showed seroconversion of anti-Env IgG and IgA antibodies, albeit the responses were weaker than in animals immunized with Env and Ribi i.m. Given that mice showed no anti-Env IgG or IgA antibodies in serum when given HIV-1 Env alone and that five i.n. immunizations with Env plus CTA1-DD adjuvant significantly enhanced anti-Env antibody responses compared with three i.n. immunizations, future studies in non-human primates should use at least four subsequent HIV-1 Env plus CTA1-DD immunizations. These experiments will also address the issue of NAbs as we did not investigate this important property of anti-Env antibodies in the present study. Nonetheless, our results from the present study clearly demonstrated that CTA1-DD should be explored further as a promising mucosal vaccine adjuvant for humoral- and cell-mediated immunity against HIV-1 Env.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agren, L. C., Ekman, L., Lowenadler, B. & Lycke, N. Y. (1997). Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J Immunol 158, 3936–3946.[Abstract]
Akhiani, A. A., Pappo, J., Kabok, Z., Schon, K., Gao, W., Franzen, L. E. & Lycke, N. (2002). Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells. J Immunol 169, 6977–6984.
Akhiani, A. A., Stensson, A., Schon, K. & Lycke, N. (2006). The nontoxic CTA1-DD adjuvant enhances protective immunity against Helicobacter pylori infection following mucosal immunization. Scand J Immunol 63, 97–105.[CrossRef][Medline]
Albrecht, H. (2003). Making sense of AIDSVAX. AIDS Clin Care 15, 42[Medline]
Andersen, C. S., Dietrich, J., Agger, E. M., Lycke, N. Y., Lovgren, K. & Andersen, P. (2007). The combined CTA1-DD/ISCOMs vector is an effective intranasal adjuvant for boosting prior Mycobacterium bovis BCG immunity to Mycobacterium tuberculosis. Infect Immun 75, 408–416.
Baldridge, J. R., McGowan, P., Evans, J. T., Cluff, C., Mossman, S., Johnson, D. & Persing, D. (2004). Taking a Toll on human disease: toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Ther 4, 1129–1138.[CrossRef][Medline]
Barnett, S. W., Lu, S., Srivastava, I., Cherpelis, S., Gettie, A., Blanchard, J., Wang, S., Mboudjeka, I., Leung, L. & other authors (2001). The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region. J Virol 75, 5526–5540.
Barnett, S. W., Srivastava, I. K., Kan, E., Zhou, F., Goodsell, A., Cristillo, A. D., Ferrai, M. G., Weiss, D. E., Letvin, N. L. & other authors (2008). Protection of macaques against vaginal SHIV challenge by systemic or mucosal and systemic vaccinations with HIV-envelope. AIDS 22, 339–348.[Medline]
Barton, G. M. & Medzhitov, R. (2002). Control of adaptive immune responses by Toll-like receptors. Curr Opin Immunol 14, 380–383.[CrossRef][Medline]
Bergmeier, L. A. & Lehner, T. (2006). Innate and adaptive mucosal immunity in protection against HIV infection. Adv Dent Res 19, 21–28.
Beutler, B. (2007). Neo-ligands for innate immune receptors and the etiology of sterile inflammatory disease. Immunol Rev 220, 113–128.[CrossRef][Medline]
Binley, J. M., Sanders, R. W., Clas, B., Schuelke, N., Master, A., Guo, Y., Kajumo, F., Anselma, D. J., Maddon, P. J. & other authors (2000). A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol 74, 627–643.
Burton, D. R., Desrosiers, R. C., Doms, R. W., Koff, W. C., Kwong, P. D., Moore, J. P., Nabel, G. J., Sodroski, J., Wilson, I. A. & Wyatt, R. T. (2004). HIV vaccine design and the neutralizing antibody problem. Nat Immunol 5, 233–236.[CrossRef][Medline]
Burton, D. R., Stanfield, R. L. & Wilson, I. A. (2005). Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci U S A 102, 14943–14948.
Choi, A. H., McNeal, M. M., Flint, J. A., Basu, M., Lycke, N. Y., Clements, J. D., Bean, J. A., Davis, H. L., McCluskie, M. J. & other authors (2002). The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant. Vaccine 20, 1733–1740.[CrossRef][Medline]
De Filette, M., Ramne, A., Birkett, A., Lycke, N., Lowenadler, B., Min Jou, W., Saelens, X. & Fiers, W. (2006). The universal influenza vaccine M2e-HBc administered intranasally in combination with the adjuvant CTA1-DD provides complete protection. Vaccine 24, 544–551.[Medline]
Derby, N. R., Kraft, Z., Kan, E., Crooks, E. T., Barnett, S. W., Srivastava, I. K., Binley, J. M. & Stamatatos, L. (2006). Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection. J Virol 80, 8745–8762.
Dickinson, B. L. & Clements, J. D. (1995). Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect Immun 63, 1617–1623.
Eliasson, D. G., Bakkouri, K. E., Schon, K., Ramne, A., Festjens, E., Lowenadler, B., Fiers, W., Saelens, X. & Lycke, N. (2008). CTA1–M2e-DD: a novel mucosal adjuvant targeted influenza vaccine. Vaccine 26, 1243–1252.[CrossRef][Medline]
Eriksson, A. M., Schon, K. M. & Lycke, N. Y. (2004). The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues. J Immunol 173, 3310–3319.
Forsell, M. N., McInerney, G. M., Dosenovic, P., Hidmark, A. S., Eriksson, C., Liljestrom, P., Grundner, C. & Karlsson Hedestam, G. B. (2007). Increased human immunodeficiency virus type 1 Env expression and antibody induction using an enhanced alphavirus vector. J Gen Virol 88, 2774–2779.
Gavin, A. L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B. & Nemazee, D. (2006). Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science 314, 1936–1938.
Girard, M. P., Bansal, G. P., Pedroza-Martins, L., Dodet, B., Mehra, V., Schito, M., Mathieson, B., Delfraissy, J. F. & Bradac, J. (2008). Mucosal immunity and HIV/AIDS vaccines Report of an International Workshop, 28–30 October 2007. Vaccine 26, 3969–3977.[CrossRef][Medline]
Grdic, D., Ekman, L., Schon, K., Lindgren, K., Mattsson, J., Magnusson, K. E., Ricciardi-Castagnoli, P. & Lycke, N. (2005). Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin. J Immunol 175, 5192–5202.
Grundner, C., Pancera, M., Kang, J. M., Koch, M., Sodroski, J. & Wyatt, R. (2004). Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins. Virology 330, 233–248.[CrossRef][Medline]
Gupta, K. & Klasse, P. J. (2006). How do viral and host factors modulate the sexual transmission of HIV? Can transmission be blocked? PLoS Med 3, e79[CrossRef][Medline]
Guy, B. (2007). The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 5, 505–517.[CrossRef][Medline]
Haase, A. T. (2005). Perils at mucosal front lines for HIV and SIV and their hosts. Nat Rev Immunol 5, 783–792.[Medline]
Harris, G., KuoLee, R. & Chen, W. (2006). Role of Toll-like receptors in health and diseases of gastrointestinal tract. World J Gastroenterol 12, 2149–2160.[Medline]
Haynes, B. F. & Shattock, R. J. (2008). Critical issues in mucosal immunity for HIV-1 vaccine development. J Allergy Clin Immunol 122, 3–9.[CrossRef]
Hemmi, H., Kaisho, T., Takeda, K. & Akira, S. (2003). The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J Immunol 170, 3059–3064.
Hladik, F. & McElrath, M. J. (2008). Setting the stage: host invasion by HIV. Nat Rev Immunol 8, 447–457.[CrossRef][Medline]
Hol, W. G. J., Sixma, T. K. & Merritt, E. A. (1995). Structure and function of E. coli heat-labile enterotoxin and cholera toxin B pentamer. In Natural Toxins: Bacterial Toxins and Virulence Factors in Disease, pp. 185–223. Edited by J. Moss, M. Vaughan, B. Iglewski & A. T. Tu. New York: Marcel Dekker, Inc.
Karlsson Hedestam, G. B., Fouchier, R. A., Phogat, S., Burton, D. R., Sodroski, J. & Wyatt, R. T. (2008). The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus. Nat Rev Microbiol 6, 143–155.[CrossRef][Medline]
Kim, M., Qiao, Z. S., Montefiori, D. C., Haynes, B. F., Reinherz, E. L. & Liao, H. X. (2005). Comparison of HIV type 1 ADA gp120 monomers versus gp140 trimers as immunogens for the induction of neutralizing antibodies. AIDS Res Hum Retroviruses 21, 58–67.[CrossRef][Medline]
Kozlowski, P. A. & Neutra, M. R. (2003). The role of mucosal immunity in prevention of HIV transmission. Curr Mol Med 3, 217–228.[CrossRef][Medline]
Kwissa, M., Kasturi, S. P. & Pulendran, B. (2007). The science of adjuvants. Expert Rev Vaccines 6, 673–684.[CrossRef][Medline]
Lavelle, E. C., Jarnicki, A., McNeela, E., Armstrong, M. E., Higgins, S. C., Leavy, O. & Mills, K. H. (2004). Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent. J Leukoc Biol 75, 756–763.
Levine, M. M. & Sztein, M. B. (2004). Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 5, 460–464.[CrossRef][Medline]
Li, Y., Svehla, K., Mathy, N. L., Voss, G., Mascola, J. R. & Wyatt, R. (2006). Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants. J Virol 80, 1414–1426.
Liang, S., Wang, M., Triantafilou, K., Triantafilou, M., Nawar, H. F., Russell, M. W., Connell, T. D. & Hajishengallis, G. (2007). The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2. J Immunol 178, 4811–4819.
Lycke, N. (2007). Mechanisms of Adjuvant Action. In Vaccine Adjuvants and Delivery Systems, pp. 53–79. Edited by M. Singh. Hoboken, NJ: John Wiley & Sons.
Maher, D., Wu, X., Schacker, T., Horbul, J. & Southern, P. (2005). HIV binding, penetration, and primary infection in human cervicovaginal tissue. Proc Natl Acad Sci U S A 102, 11504–11509.
Marks, E., Verolin, M., Stensson, A. & Lycke, N. (2007). Differential CD28 and inducible costimulatory molecule signaling requirements for protective CD4+ T-cell-mediated immunity against genital tract Chlamydia trachomatis infection. Infect Immun 75, 4638–4647.
Mascola, J. R. (2003). Defining the protective antibody response for HIV-1. Curr Mol Med 3, 209–216.[CrossRef][Medline]
Mazanec, M. B., Kaetzel, C. S., Lamm, M. E., Fletcher, D. & Nedrud, J. G. (1992). Intracellular neutralization of virus by immunoglobulin A antibodies. Proc Natl Acad Sci U S A 89, 6901–6905.
Mazzoli, S., Trabattoni, D., Lo Caputo, S., Piconi, S., Ble, C., Meacci, F., Ruzzante, S., Salvi, A., Semplici, F. & other authors (1997). HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals. Nat Med 3, 1250–1257.[CrossRef][Medline]
McGettrick, A. F. & O'Neill, L. A. (2007). Toll-like receptors: key activators of leucocytes and regulator of haematopoiesis. Br J Haematol 139, 185–193.[CrossRef][Medline]
McNeal, M. M., Basu, M., Bean, J. A., Clements, J. D., Lycke, N. Y., Ramne, A., Lowenadler, B., Choi, A. H. & Ward, R. L. (2007). Intrarectal immunization of mice with VP6 and either LT(R192G) or CTA1-DD as adjuvant protects against fecal rotavirus shedding after EDIM challenge. Vaccine 25, 6224–6231.[CrossRef][Medline]
Mestecky, J., Moldoveanu, Z. & Russell, M. W. (2005). Immunologic uniqueness of the genital tract: challenge for vaccine development. Am J Reprod Immunol 53, 208–214.[CrossRef][Medline]
Montefiori, D., Sattentau, Q., Flores, J., Esparza, J. & Mascola, J. (2007). Antibody-based HIV-1 vaccines: recent developments and future directions. PLoS Med 4, e348[CrossRef][Medline]
Mutsch, M., Zhou, W., Rhodes, P., Bopp, M., Chen, R. T., Linder, T., Spyr, C. & Steffen, R. (2004). Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland. N Engl J Med 350, 896–903.
Neutra, M. R. & Kozlowski, P. A. (2006). Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6, 148–158.[CrossRef][Medline]
Parren, P. W., Marx, P. A., Hessell, A. J., Luckay, A., Harouse, J., Cheng-Mayer, C., Moore, J. P. & Burton, D. R. (2001). Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 75, 8340–8347.
Pasare, C. & Medzhitov, R. (2005). Control of B-cell responses by Toll-like receptors. Nature 438, 364–368.[CrossRef][Medline]
Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., Martin, M., van Griensven, F., Hu, D., Tappero, J. W. & Choopanya, K. (2006). Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis 194, 1661–1671.[CrossRef][Medline]
Sanders, R. W., Vesanen, M., Schuelke, N., Master, A., Schiffner, L., Kalyanaraman, R., Paluch, M., Berkhout, B., Maddon, P. J. & other authors (2002). Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol 76, 8875–8889.
Shattock, R. J., Haynes, B. F., Pulendran, B., Flores, J. & Esparza, J. (2008). Improving defences at the portal of HIV entry: mucosal and innate immunity. PLoS Med 5, e81[CrossRef][Medline]
Simmons, C. P., Mastroeni, P., Fowler, R., Ghaem-maghami, M., Lycke, N., Pizza, M., Rappuoli, R. & Dougan, G. (1999). MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants. J Immunol 163, 6502–6510.
Sin, J. I., Kim, J. J., Boyer, J. D., Ciccarelli, R. B., Higgins, T. J. & Weiner, D. B. (1999). In vivo modulation of vaccine-induced immune responses toward a Th1 phenotype increases potency and vaccine effectiveness in a herpes simplex virus type 2 mouse model. J Virol 73, 501–509.
Srivastava, I. K., VanDorsten, K., Vojtech, L., Barnett, S. W. & Stamatatos, L. (2003). Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region. J Virol 77, 2310–2320.
Stephenson, I., Zambon, M. C., Rudin, A., Colegate, A., Podda, A., Bugarini, R., Del Giudice, G., Minutello, A., Bonnington, S. & other authors (2006). Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers. J Virol 80, 4962–4970.
Tritto, E., Muzzi, A., Pesce, I., Monaci, E., Nuti, S., Galli, G., Wack, A., Rappuoli, R., Hussell, T. & De Gregorio, E. (2007). The acquired immune response to the mucosal adjuvant LTK63 imprints the mouse lung with a protective signature. J Immunol 179, 5346–5357.
Underhill, D. M. & Ozinsky, A. (2002). Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 14, 103–110.[CrossRef][Medline]
Vajdy, M., Singh, M., Kazzaz, J., Soenawan, E., Ugozzoli, M., Zhou, F., Srivastava, I., Bin, Q., Barnett, S. & other authors (2004). Mucosal and systemic anti-HIV responses in rhesus macaques following combinations of intranasal and parenteral immunizations. AIDS Res Hum Retroviruses 20, 1269–1281.[CrossRef][Medline]
Veazey, R. S., Shattock, R. J., Pope, M., Kirijan, J. C., Jones, J., Hu, Q., Ketas, T., Marx, P. A., Klasse, P. J. & other authors (2003). Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 9, 343–346.[CrossRef][Medline]
Vogel, F. R. (1995). Immunologic adjuvants for modern vaccine formulations. Ann N Y Acad Sci 754, 153–160.[Medline]
Wicker, S., Rabenau, H. F., Gottschalk, R., Doerr, H. W. & Allwinn, R. (2007). Seroprevalence of vaccine preventable and blood transmissible viral infections (measles, mumps, rubella, polio, HBV, HCV and HIV) in medical students. Med Microbiol Immunol 196, 145–150.[CrossRef][Medline]
Xu, R., Srivastava, I. K., Greer, C. E., Zarkikh, I., Kraft, Z., Kuller, L., Polo, J. M., Barnett, S. W. & Stamatatos, L. (2006a). Characterization of immune responses elicited in macaques immunized sequentially with chimeric VEE/SIN alphavirus replicon particles expressing SIVGag and/or HIVEnv and with recombinant HIVgp140Env protein. AIDS Res Hum Retroviruses 22, 1022–1030.[CrossRef][Medline]
Xu, R., Srivastava, I. K., Kuller, L., Zarkikh, I., Kraft, Z., Fagrouch, Z., Letvin, N. L., Heeney, J. L., Barnett, S. W. & Stamatatos, L. (2006b). Immunization with HIV-1 SF162-derived Envelope gp140 proteins does not protect macaques from heterologous simian-human immunodeficiency virus SHIV89.6P infection. Virology 349, 276–289.[CrossRef][Medline]
Yang, X., Florin, L., Farzan, M., Kolchinsky, P., Kwong, P. D., Sodroski, J. & Wyatt, R. (2000). Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution. J Virol 74, 4746–4754.
Yang, X., Wyatt, R. & Sodroski, J. (2001). Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers. J Virol 75, 1165–1171.
Yang, X., Lee, J., Mahony, E. M., Kwong, P. D., Wyatt, R. & Sodroski, J. (2002). Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin. J Virol 76, 4634–4642.
Zignol, M., Peracchi, M., Tridello, G., Pillon, M., Fregonese, F., D'Elia, R., Zanesco, L. & Cesaro, S. (2004). Assessment of humoral immunity to poliomyelitis, tetanus, hepatitis B, measles, rubella, and mumps in children after chemotherapy. Cancer 101, 635–641.[CrossRef][Medline]
Received 8 July 2008;
accepted 22 August 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
