It is postulated that unique nanoscale proteomic features of immunogen on vaccine particles may determine immunogen-packing density, stability, specificity, and pH-sensitivity on the vaccine particle surface and thus impact the vaccine-elicited immune responses. To test this presumption, we employed near-filed scanning optical microscopy (NSOM)- and atomic force microscopy (AFM)-based nanotechnology to study nano-structural and single-molecule force bases of Yersinia pestis (Y. pestis) V immunogen fused with protein anchor (V-PA) loaded on gram positive enhancer matrix (GEM) vaccine particles. Surprisingly, the single-molecule sensitive NSOM revealed that approximately 90% of V-PA immunogen molecules were packed as high-density nanoclusters on GEM particle. AFM-based single-molecule force analyses indicated a highly stable and specific binding between V-PA and GEM at the physiological pH. In contrast, this specific binding was mostly abrogated at the acidic pH equivalent to the biochemical pH in phagolysosomes of antigen-presenting-cells in which immunogen protein is processed for antigen presentation. Intranasal mucosal vaccination of mice with such immunogen loaded on vaccine particles elicited robust antigen-specific immune response. This study indicated that high-density, high-stability, specific, and immunological pH-responsive loading of immunogen nanoclusters on vaccine particles could readily be presented to the immune system for induction of strong antigen-specific immune responses.