Although available recombinant Hepatitis B vaccines are efficiently applied in the prevention of Hepatitis B virus (HBV) infection, but no therapeutically efficacious vaccine is introduced for the clearance of HBV infection in the carriers and chronically infected patients. An ideal HBV vaccine would be both preventive and therapeutic which means that this vaccine should promote the production of specific neutralizing antibodies and cell-mediated responses (particularly T helper 1 and cytotoxic T-cells) against the virus. One of the main obstacles in the procedure of such a vaccine production is the lack of suitable adjuvants with cellular responses inducing features. The classical adjuvant for vaccines, and until recently the only FDA-approved adjuvant, is alum which is used in the commercially available HBV vaccines ⁽¹⁾. Alum provides a particle upon which the vaccine is precipitated. Although precipitation onto alum promotes uptake of the immunogen, alum is a poor activator of DCs and does not induce the production of IL-12 ⁽²⁾. As a result, vaccines containing alum initiate a Th2-type antibody response ⁽³⁾.

Complete Freund's adjuvant (CFA) is another powerful adjuvant consisting of inactivated Bacillus Calmette-Guerin (BCG) and a mixture of different TLR ligands in a mineral oil solution ⁽¹⁾. Although BCG is a strong activator of DCs and induces a Th1 response, CFA is not approved for human use ⁽¹⁾. Application of adjuvants with microbial origins is a recently highlighted approach in the vaccinology trials ⁽⁴⁾. Archaeosomes are among these microbial compounds with both adjuvant and liposomal activities and features ⁽³⁾. Archaeosomes are liposomes made of polar lipids obtained from archaeal (archaebacterial) cells. The mammalian-like archaeal lipids consist of isoprenoid chain glycerolipids. Archaeol (di-O-phytanylglycerol) is double ether of sn-1-glycerol where positions 2 and 3 are bound to phytanyl residues. The archaeols are Archaea homologs of Diacylglycerols (DAGs) ⁽⁵⁾. In certain archaea such as Methanobrevibacter smithii (M. Smithii), archaeol is found in the dimer form namely caldarchaeol which enhances the stability of archaeosomes originated from this archaea ⁽⁶⁾.

Some immunologically important features of archaeosomes include high stability of archaeal lipids as building blocks of archaeosomes which leads to long-lasting memory of immune responses, immunopotent interaction with Antigen Presenting Cells (APC) and most importantly shifting and leading the antigen processing pathway to MHC class I mechanisms and subsequently intensifying CD8⁺ T-cell responses ^{(3, 5)}. Available vaccines are mostly designed according to the immunostimulatory properties of Hepatitis B surface Antigen (HBsAg) which is also called envelope antigen. The HBV genome encodes for three related envelope proteins termed L, M, and S. The three envelope proteins are produced from a single Open Reading Frame (ORF) through alternative translation start sites. HBsAg is an antigenically heterogeneous protein, with a common antigen designated a, and two pairs of mutually exclusive antigens, d and y, and w and r, resulting in 4 major subtypes, namely adw, ayw, adr and ayr ⁽⁷⁾.

In the present study, recombinant HBsAg (Recombinant HBV HBsAg Antigen, Subtype adw, Acrobiosystems, USA) was encapsulated into M. smithii archaeosomes. Balb/c mice were immunized with this compound and humoral and cytokine secretion patterns of immunized models were analyzed.

HBV commercial vaccines mostly promote efficient humoral responses for prevention of infection. A considerable concern in HBV vaccine researches is the lack of a therapeutic vaccine for clearance of infection through the augmentation of T helper 1 and CD8⁺ T-cells ⁽⁹⁾. Lack of an applicable adjuvant to be formulated in the therapeutic HBV vaccine candidates has been a consistent problem in vaccine researches. Most of known adjuvants are not desirable for human use because of undesirable reactions or their weak effect on the induction of cellular responses ⁽¹⁰⁾. Archaeal liposomes (archaeosomes) are among newly introduced adjuvants with potency to augment humoral and cell-mediated responses, particularly immune responses with the dominance of T-helper 1 and CD8⁺ T-cells ⁽¹¹⁾.

In the present study, an attempt was made to evaluate adjuvant properties of M. smithii archaeosomes to induce humoral and cellular responses by the assessment of IgG (total and subclass) and cytokine responses, respectively. Total anti HBs IgG and related subclasses were analyzed by ELISA and all indicated the potency of HBsAg-containing archaeosomes to induce significant IgG responses against HBsAg in comparison to other groups immunized with HBsAg and HBsAg⁺C/IFA (p< 0.05). The advantage of archaeosomes to induce strong humoral responses to entrapped antigen has been previously confirmed for varied antigens (hen egg lysozyme, ovalbumin, cholera toxin) by various immunization routes ^{(3, 12)}. IgG subclasses analysis verified the dominance of IgG2a in comparison to IgG1 in the HBsAg-containing archaeosome immunized mice as a sign of T-helper 1 orientation of cell-mediated responses since supremacy of IgG2a and IgG1 is commonly considered as the dominance of T-helper 1 and T-helper 2 sub-population responses, respectively ⁽¹³⁾. The higher ratio of IgG2a/total IgG titer (0.71) in comparison with the ratio of IgG1/total IgG titer (0.33) in the HBsAg-containing archaeosome immunized mice sera is another sign of supremacy of T-helper 1 responses. Dominance of T-helper 1 oriented responses against viral infections would be a promising achievement in the application of archaeal adjuvants in the development of a therapeutic HBV vaccine.

The antibody secretion pattern was verified by the ELISpot results since the frequency of IFN-γ secreting splenocytes was significantly higher than IL-4 SFCs following the immunization with HBsAg-containing archaeosomes. IFN-γ secreting splenocytes in the group immunized by HBsAg-containing archaeosomes were also significantly higher in comparison to the groups immunized by HBs-Ag and HBsAg formulated in Freund's adjuvant (p< 0.05).

Similar to the results of IgG subclasses analysis that indicated the ratio of IgG2b/total IgG titer as the highest ratio between all subclasses, the ratio of IFN-γ/IL-4 SFCs was also the highest one in the group immunized by HBsAg-archaeal adjuvant as an indication of T-helper 1 oriented responses. According to the ability of M. smithii archaeosomes in the enhancement of MHC class I antigen presentation and the supremacy of IgG2a subtype and IFN-γ in the mentioned group, CTLs and T-helper 1 cells responses were suggested as the cellular responses induced by this administered formulation ⁽¹⁴⁾.

Although Hepatitis B vaccine is not a vague concept now, at least in the prevention of infection, findings of the present study would indicate the potency of M. smithii archaeosomes to induce strong T-helper 1 and IFN-γ mediated responses against HBsAg as a model of a viral antigen. However, there is also a clinical need for a therapeutically effective vaccine for Hepatitis B that induces robust cell-mediated response capable of viral clearance in the chronic infection. There are evidences indicating the impairment of T-helper 1 responses during the HBV infection leading to viral immune escape and consequently chronic HBV infection ⁽¹⁵⁾. The immunopotency of HBsAg-containing archaeosomes in the induction of T-helper 1 responses and IFN-γ production would be also therapeutically efficient to compensate T-helper 1 cells deficiency during HBV infection. Probable mechanism for enhancement of cellular immunity by this formulation is related to augmentation of immunological interactions and recruitment of antigens via macrophages and other APC ⁽¹¹⁾.

Other efforts for development of a therapeutic HBV vaccine have been made previously. Chuai et al evaluated the effect of several adjuvants including alum, CpG and poly-riboinosinic polyribocytidylic acid [poly(I:C)] to enhance anti-HBV response when boosted with recombinant adenoviral vector vaccine. Among the adjuvants tested, CpG/alum and poly(I:C)/alum combinations induced the specific production of high antibody titres with a Th1bias. In comparison to the present study, the frequency of IFN-γ SFCs following their immunization was considerably less than the frequency induced by HBsAg-containing archaeosome immunization. D. Morrey et al applied a Cationic Lipid DNA Complex (CLDC) as an adjuvant for HBsAg. Their combination efficiently induced both B and T-cells responses and they suggested that CLDC can be a promising adjuvant for therapeutic HBV vaccines although the orientation of the cellular responses was not specified in their study since IFN-γ was the only assayed cytokine.

In contrast, the present study assayed IFN-γ and IL-4 as an index for T helper 1 and T helper 2 responses, respectively. Buchmann et al evaluated the potential of a formulation comprising particulate HBsAg and core antigen (HBcAg), and the saponin-based ISCO-MATRIX™ adjuvant for its ability to stimulate T and B cell responses in C57BL/6 mice. Their candidate vaccine induced strong CD8⁺ T-cell responses and efficiently reduced HBc-Ag expression in the liver of immunized mice. Application of archaeosomes in combination with different antigens is a vastly studied concept in vaccine researches. Priming and boosting with Bovine Serum Albumin (BSA) entrapped in archaeosomes were shown to induce significantly higher antibody titers in comparison to immunization with the same antigen entrapped in conventional liposomes ⁽¹⁶⁾.

The advantage of archaeosomes to induce strong humoral responses to entrapped antigen has been confirmed for various antigens (hen egg lysozyme, ovalbumin, cholera toxin), and by multiple immunization routes (IP, IM, SC) and in murine strains of different genetic background (C3H/HeJ, C57BL/6, BALB/c). Similar to the results of the current study, analyzing the antibody isotype distribution in mice immunized with antigen in varied archaeosomes, indicated induction of strong IgG1, IgG2a and IgG2b titres, in contrast to alum that evokes little IgG2a isotype switching ⁽³⁾. Archaeosomes originated from certain archaea such as M.smithii and Thermoplasma acidophilum are more stable due to high percentage of caldarchaeols in their structures ⁽¹⁷⁾. This type of archaeosomes limits phagolysosomal antigen processing and enhances MHC class I presentation ⁽¹⁸⁾. This would be potentially beneficial in the induction of MHC class I and CTL responses against intracellular and viral infections. These responses are particularly favorable in the clearance of infected hepatocytes during HBV infection ⁽¹⁹⁾.

According to the importance of cellular responses along with specific antibody reactions in all vaccinology aspects, the characteristic of elicited responses is not only applicable in HBV vaccine researches but also would be considered in other vaccine researches as well.