Neutralizing antibodies and cytotoxic T cells play certainly an important role in eradicating or containing viral infection but they represent the “tip of the iceberg” of the arsenal potential of the immune system. Because HIV-1 is mainly acquired at mucosal sites, mucosal immune responses that interfere with HIV-1 attachment and migration across the mucosa, and promote viral clearance may contribute also to prevent sexual HIV-1 transmission. Protective mucosal antibodies could be elicited in the female genital tract by vaginal vaccination but this administration route poses several challenges and may require a mucosal adjuvant in order to induce an optimal immune response 
We have designed a prophylactic HIV-1 vaccine candidate that is potentially capable of protecting the initial sites of viral entry, especially the female genitals and the rectum, by inducing a mucosal humoral immune response without the need of local vaccination. The proposed MYM-V101 candidate HIV-1 vaccine was tested in a Phase I “Proof of Principle” to evaluate its safety and tolerability, when administered via intramuscular and intranasal routes in healthy young women.
The rationale for this immunization regimen was based on the postulate that intranasal priming may not be very efficient unless a potent mucosal adjuvant is added to the formulation. However, vaccination by intranasal route without adjuvant could work more efficiently if applied as a boost, following adequate priming via the intramuscular route. Although such immunization regimen may be less practical for medical care providers, it may minimize safety and regulatory concerns if mucosal adjuvant is absent from the vaccine. MYM-V101 can be considered as a safe vaccine that is well tolerated, when administered by the intramuscular () and intranasal () routes at the tested concentrations. No significant difference could be observed between the safety results of vaccinated groups and placebo recipients.
Very few HIV trials have tried without success to monitor mucosal antibodies during prophylactic vaccination 
. We may postulate that the detection assays employed at that time were not sensitive and robust enough for quantifying very low levels of specific mucosal antibodies. For our trial, we have developed the Imperacer®, a fast, robust and ultrasensitive detection assay 
already used by pharmaceutical industries for other applications, which is suitable for detecting specific low mucosal antibody concentrations in the range of pg to ng/mL. The assay is fundamentally based on ELISA but the antibody-enzyme conjugates are replaced by antibody-DNA conjugates, which can be amplified by PCR 
. This technique was validated during clinical development and it allows reliable detection of antibodies in serum and mucosal samples, offering also the main advantage of consuming few μL.
The vaccine MYM-V101 has induced P1-specific IgG and IgA responses in serum already within 4 weeks after the first vaccination (). There is a clear benefit of the first two intramuscular injections for triggering the systemic humoral response. The third vaccine dose given intranasally elicited only a significant antibody boost of serum IgGs in the HD group. No clear effect of the third dose was seen on serum IgA levels and no booster effect of the fourth dose could be demonstrated. Currently, it cannot be excluded that the third and fourth vaccine doses may have a beneficial effect by improving the affinity and/or the antiviral capacity of the elicited antibodies, as well as improving the memory response. However, this needs to be investigated further.
One month after the last vaccination, vaginal and rectal P1-specific IgGs were detected in almost all vaccinees. The P1-specific IgGs found in mucosal samples may be strictly derived from local production but can also partially have a circulatory origin. Due to unexpected high pre-immune vaginal reactivity toward the P1 antigen (high pre-immune ΔCT value) observed in some subjects, the analysis per group did not reveal a significant increase in mucosal P1-specific IgAs in immune samples. Unspecific binding is always possible but the observed reactivity could also be due to specific binding detected by the ultrasensitive Imperacer® technique. We may postulate that pre-immune sample reactivity could be related to: i) The presence of auto-antibodies against self proteins that cross-react with HIV-1 gp41 motifs, since HIV-1 is known to share several human protein homologies 
; or ii) Subjects were exposed to HIV-infected individuals prior to this study and have developed specific mucosal anti-gp41 antibodies, while remaining seronegative, as reported in HIV exposed-seronegative (HESN) individuals 
. The latter hypothesis is very unlikely, considering the “profile” of our volunteers but we cannot exclude this possibility. More investigations would be needed to further explore these possibilities but it was not the objective of this exploratory study conducted on limited number of subjects.
When ΔCT values of immune samples were individually considered and compared to their corresponding pre-immune ΔCT values, many subjects taken separately had a ΔΔCT value (ΔCT immune – ΔCT pre-immune) above the 1.966 cut-off, suggesting a net increase of mucosal P1-specific IgAs after vaccination with MYM-V101 (see ). P1-specific IgAs were detected in vaginal samples of 63% (LD) and 43% (HD) of the subjects with at least one positive mucosal sample out of three. We have noticed that detection of vaginal P1-specific IgAs seems to pose a big challenge due to the variation that occurs over time within a single individual. This may be explained in part by the hormonal and menstrual cycle fluctuations already reported 
, which could influence the amount of IgAs already low in the women vagina, while IgAs in the endocervix are more abundant and easier to detect. The low level of IgAs, respective to IgGs was expected in the vaginal secretion but the low frequency of positive samples may also point towards a technical problem, as IgA is the dominant antibody isotype in the lower intestinal tract. The current antibody detection methodology and rectal harvesting technique will require further optimization for optimal IgA detection.
To alleviate the study burden for the participants, the collection of mucosal samples on week 9 (one week after the second intramuscular injection) was abandoned. Therefore, the benefit of the third injection (first intranasal administration) on mucosal antibodies is difficult to appreciate at this stage. Meanwhile, it is known that intranasal vaccination may solicit distant mucosal and systemic immune responses 
, depending on the induction of specific sets of homing receptors during the interaction on T and B cells with mucosal dendritic cells 
. However, activation of the mucosal responses at the vaginal and intestinal levels by the intramuscular route, using non-replicative vectors like virosomes might be more an exception than the rule. In an upcoming clinical trial, additional mucosal samples will be collected to clarify the respective contribution of the intramuscular and intranasal route for the induction of mucosal immunity. The available data allows us to conclude that MYM-V101 has successfully induced specific mucosal antibodies.
At the end of the study (week 29, and ), the mean total P1-specific antibody concentration in vaginal samples (LD: 6.18 ng/mL and HD: 1.6 ng/mL) is at least 100-fold lower than serum samples (LD: 658 ng/mL and HD: 410 ng/mL). These low mucosal antibody concentrations preclude their successful testing in current in vitr
o neutralization assays, which were initially developed for serum samples and generally require at least 200 ng/well of neutralizing specific antibodies 
. Currently, we cannot exclude the presence of low levels of neutralizing antibodies in vaginal or rectal fluids and antibody purification might be necessary for detecting them. Meanwhile, adding purification steps for specific IgGs and IgAs could introduce risks of losing or affecting the quality of samples and this approach might not be easily applicable to clinical trials evaluating thousands of samples. It might be more realistic to improve the in vitro
neutralization assay sensitivity by at least 100-fold, ideally requiring less than 1 ng/well of specific antibodies for reliable testing of low amount of unpurified mucosal samples.
Although few ng/mL of specific mucosal antibodies might be perceived as a very low antibody concentration in the vaccine field, respective to serums that generally contain μg/mL of specific antibodies, it still represents billions of antibodies per mL of vaginal secretion. Furthermore, it is likely an underestimation, considering the contribution of the mucosal antibodies also trapped in the mucus or located in the lamina propria underneath the mucosal tissue that could not be estimated from the collected secretion samples of this study.
Transcytosis inhibition was investigated only for vaginal samples, as our previous studies had shown that circulatory antibodies could not inhibit HIV-1 transcytosis 
and rectal samples were too limited. Although the transcytosis assay has been standardized to some extent in various laboratories, variations are inherent to in vitro
cell-based assays and very often, experiments are conducted at least twice for confirming the in vitro
observations. Transcytosis assay was developed to obtain robust data (qualitative observation) but was not formally qualified, as it is done for bio-analysis of the active pharmaceutical ingredient. Acting as a candidate biomarker for induction of functional mucosal immunity, transcytosis assay may provide reliable data for detecting antiviral activities, such as antibodies interfering with HIV-1 passage across in vitro
cell monolayer, mimicking the mucosal epithelium found in the endocervix or intestinal tract. Eighty percent of the vaginal samples were inhibiting HIV-1 transcytosis (), as opposed to placebo samples that had no activity. For some mucosal samples, discrepancies were found between the observed transcytosis inhibition that was shown to be mainly dependent on IgAs 
and the absence of mucosal IgAs detection. We are postulating that the current antibody detection assay may not detect the full IgA antibody repertoire and further optimization is required. IgGs could also act in synergy with IgAs for optimal antiviral activities for blocking early steps of HIV transfer and infection at the mucosal sites 
. We postulate that a prophylactic HIV-1 vaccine as MYM-V101 that elicits mucosal anti-gp41 antibodies in the range of 5–10 ng/mL or more (at least 1010
molecules of antibodies/mL of secretion) could efficiently block mucosal HIV-1 acquisition from semen of HIV-1 acutely infected men, which generally contains only 102
cell-free infectious particles or thousands of HIV-infected cells 
The mucosal immune system of the male reproductive tract in human 
and macaque 
has been studied only recently. These studies have revealed the presence of antibodies but also of HIV-1 target cells like dendritic cells, Langerhans cells and T lymphocytes, explaining why the penile foreskin, inner foreskin mucosal epithelium, glans and urethra, are potential sites of HIV-1 acquisition in men. These observations are suggesting that vaccines eliciting mucosal immunity reaching the male reproductive tract could protect men from acquiring HIV. In subsequent human trials, the vaccine safety and immunogenicity will also be tested in men, in parallel to women for monitoring vaccine-induced antibodies in the genital tract of both genders.
Virus-like particles (VLPs) and enveloped VLPs such as virosomes harbour antigens at their surface, which are seen as repetitive motifs that are efficiently recognized by B cell surface antibodies, leading to their activation. Most VLP-based vaccines are employing adjuvant such as alum salts and Toll-like receptor agonists, while influenza-virosomes as enveloped VLPs are used as stand-alone products. Therefore, comparing the immune responses induced by both adjuvanted VLP and virosome is difficult, especially if pre-existing immunity impact the vaccine-induce immune response. Most people have natural pre-existing immunity against influenza, comprizing both humoral and cellular immunity (CD4+
T cells), which can be beneficial to vaccination with influenza-virosomes 
. Influenza-specific antibodies were shown to bind to virosomes and facilitate their delivery inside the endosomes of antigen presenting cells, favouring a Th2 response characterized by a robust antibody production, as observed in our Phase I study.
Antigen cross-presentation 
leading to CTL induction is also possible with virosomes. However, the immunodominant human CTL epitopes ERYLKDQQL and CSGKLIC in the HXB2 gp41 ectodomain (HIV gp41 CTL epitope data base) are absent from our P1 peptide used for vaccination, which renders unlikely the induction of CD8+
T cell response. Meanwhile, it was important to verify this aspect and in vitro
stimulation of PBMC with P1 peptide followed by intracellular cytokine staining was unable to reveal P1-specific CD4+
Th1 response, although the threshold sensitivity of this method might be too weak for detecting low level of cell-mediated response.
Both IgG or IgA antibodies may bind to their respective Fc receptor 
and trigger various viral clearance mechanisms through Fc-mediated effector functions: Complement activation 
, antibody-mediated phagocytosis 
, or engagement of antibodies with NK cells, neutrophils or macrophages that leads to ADCC 
. The potential protective role of ADCC induced by prophylactic vaccination was already reported in the vaginal tract of vaccinated NHP with virosome-gp41 
and serum of vaccinated subjects with ALVAC and AIDSVAX that developed antibodies toward the gp120 
. Presence of HIV specific mucosal antibodies capable of ADCC activity were also shown to reduce risk of vertical transmission to breastfed infants of HIV-1 positive women 
. Therefore, ADCC activity may represent an important arm of the immune defense against HIV-1. Due to insufficient material, such binding non-neutralizing antibodies with ADCC activity could not be tested in this Phase I study but they will be part of future clinical investigations.
The idea of inducing IgAs as important and complementary players with IgGs in mucosal and systemic protection represents a new avenue in the HIV-1 vaccine field. It will be interesting to determine in the future if blood and mucosal antibodies toward the same antigen have different or similar epitope specificities, antibody isotype and how this may impact the antiviral functions in the systemic and mucosal immune compartments.
This Phase I represents the “Proof of Principle” that it is possible to elicit both specific IgGs and IgAs in circulation, as well as in vaginal and intestinal mucosal tissues, using virosome-based vaccines. The current study did not allow a broad coverage of potential antiviral activities. With recent in vitro
immune assay developments, a spectrum of innovative immuno-monitoring investigations will be explored and future clinical trials will benefit from cutting edge ultrasensitive antibody detection assays combined with new functional antibody assays for evaluating various antiviral activities. These investigations will provide new insights regarding protective immune mechanisms from blood and/or mucosal compartments, as well as new potential surrogate markers of protection. In this study, the presence of vaccine-induced mucosal anti-gp41 antibodies with antiviral activities has confirmed the previous results in NHP 
, which further supports the approach of gp41-virosome as a promising vaccine strategy to induce mucosal antibodies for reducing sexually-transmitted HIV-1.