Previous studies have demonstrated the production of AFCo1 on a lab scale and preliminary work at higher scale demonstrated that CFS could be used to obtain larger amounts of cochleates [4
In the present work, the efficiency of the immune response of the AFCo1 obtained by tangential diafiltration (pilot scale) was demonstrated. The results suggest that the i.n route is able to induce significant and antigen specific IgA levels in saliva and of IgG in serum after immunization of BALB/c mice with 3 dose of AFCo1. High titers of IgG1 and IgG2a in serum were also induced with i.n administration of AFCo1. The i.n administration with AFCo1 generated a Th1 polarized pattern, characterized by high levels of IgG2a and induction of IFNγ. This agrees with several reports that suggest that nasal associated lymphoid tissue is a place where the induction of Th1 response is favored [7
]. In addition, the immunogenicity and pattern induction of AFCo1 obtained by CFS corresponds with previous results obtained with AFCo1 obtained by the lab scale dialysis rotary method [2
In this study we also evaluated the capability of splenocytes obtained from the immunized animals to be stimulated in vitro
to produce IFNγ. The AFCo1 efficiently stimulate this cytokine suggesting activation and differentiation of the CD4 lymphocytes (LΦT) into Th1 cells. Considering that this cytokine is produced principally by the LΦT, then IFNγ might be produced by Th1 type cells isolated from spleen [9
We explored the adjuvant properties of AFCo1 with addmixed heterologous antigens (OVA or PepVacTB1) via the i.n route. The results showed that i.n immnunization with AFCo1+OVA or AFCo1+PepVacTB1 induced high levels of anti OVA or PepVacTB1 IgG, IgG1 and IgG2a antibodies in sera and specific IgA in saliva. We have demonstrated the adjuvant capability of the AFCo1 obtained by CFS when co-administered with heterologous antigens by the i.n route. Previous studies have shown i.n immunization with AFCo1 obtained by the dialysis method has adjuvant effects on co-administered heterologous antigens such as OVA [8
In addition, stability studies of AFCo1 were satisfactorily concluded in order to develop safe formulations for nasal application. Pharmaceutical and preclinical evaluation of new adjuvants, as part of vaccine formulations, is relevant in their efficacy and safety evaluation. Detailed information about the preclinical experiments with AFCo1 were previously published by Infante et al.
]. These authors demonstrate that AFCo1 successfully passed the tests of local tolerance and preclinical toxicity (single and repeated doses). A single dose was classified as non irritating locally and showed no systemic toxicity, so AFCo1 is considered potentially non toxic for intranasal administration to humans in this experiments. Repeated doses experiments, which included: complete blood count, biochemical and histopathological tests, showed that AFCo1 was rated as non-irritating locally and with no evidence of systemic toxicity, so it was considered potentially non toxic to humans by intranasal administration in repeated doses [10
New vaccines containing new adjuvants represent a challenge for regulatory authorities, which are actively discussing standardization of guidelines to evaluate and develop adjuvants in the near future [11
In summary, the present study demonstrates the efficacy of AFCo1 obtained on pilot scale through the crossflow system. AFCo1 obtained by this methodology produced high level of specific antibodies in serum and saliva by the mucosal route and have the capacity to enhance the mucosal response against Neisseria antigens in AFCo1 using OVA or PepVacTB1 as model antigens. Tangential filtration would ensure the production of a large scale adjuvant that is safe and immunogenic.