For negative control, animals received 100 l PBS in MPL/DDA at each immunization. AC02 showed similar affinity to high-mannose specific SSR240612 mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02. == Conclusion == These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common SSR240612 practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines. Keywords:HIV-1 vaccine, HIV-1 envelope, signal peptide, glycosylation, antigenicity, immunogenicity, antibody-dependent cellular phagocytosis, neutralization == Introduction == The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is the key target for the development of HIV-1 vaccines. The HIV-1 Env is synthesized as a gp160 precursor protein in the endoplasmic reticulum (ER), where signal peptide (SP) cleavage, folding, addition of high-mannose glycans, and trimerization, in association with molecular chaperones, takes place (14). Once the nascent polypeptide attains its native folding state the SP is cleaved, and the Env egresses from the ER and translocates to the Golgi apparatus (1,511). In the Golgi, gp160 is cleaved by host furin-like proteases to generate a transmembrane gp41 subunit and a non-covalently associated surface gp120 subunit. Three gp120-gp41 heterodimers assemble to form the trimeric functional Env spikes that are then directed to the plasma membrane for incorporation into virions (1,911). The gp120 subunit is the primary viral component that is exposed on the virion surface and is the key target for neutralizing antibodies (12). The HIV-1 Env has evolved a variety of mechanisms to evade host antibody (Ab) responses, including tolerance to escape mutations in immunogenic epitopes (13,14), extensive glycosylation (1518), conformational masking (19), mimicry of SSR240612 host proteins (20), and low expression of Env on virus-infected cells and virions (21,22). Together, these features have posed substantial barriers to the development of effective Env-based vaccines. Of the many clinical vaccine trials conducted to date, RV144 is the only one that showed a modest efficacy, 59.9% at 1 year and 31.2% at 3.5 years post-vaccination (2325). The trial did not elicit broadly neutralizing antibodies (bNAbs); rather, non-neutralizing Abs (nNAbs) against the V1V2 and Ab-dependent Fc-effector functions were identified as the correlates of protection (2630). Epitopes of the V1V2 Abs have been grouped into at least three types: V2i, V2p, and V2q (31,32). RV144 vaccinees produced V2p- and V2i-type Abs, but not the V2q-type Abs CX3CL1 (24,28). The trial tested the prime-boost combination of two vaccines: ALVACHIV vaccine (prime) and AIDSVAXB/E vaccine (boost). The ALVAC-HIV utilized a recombinant canarypox vector to express SSR240612 HIV-1 LAI Gag and Pol alongside monomeric 92TH023 gp120 linked to the transmembrane anchoring portion of LAI gp41. The SP of HIV-1 Env is known to be responsible for poor expression and low secretion of Env (33). To achieve higher expression of Env protein immunogens, replacing the Env SP with non-HIV-1 SPs is a common strategy.