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Here, we create and evaluate rVSV vaccines for Machupo virus (MACV), a highly pathogenic New World (NW) mammarenavirus that causes Bolivian hemorrhagic fever. VSV genome rearrangements impact attenuation of the viral vector and the immune response to the vaccine. Therefore, in this study, we directly compare the effect of altering the MACV glycoprotein complex (GPC) position in the VSV genome on humoral and cellular immune responses to vaccination by evaluating replication, antigen expression, and immunogenicity of rVSVs expressing MACV GPC in the first position of the genome (rVSV-MACV1) and in the traditional glycoprotein gene position (rVSV-MACV5). rVSV-MACV1 expresses higher levels of MACV GPC in infected cells and in virions, but the increased antigen does not translate to a more robust immune response. rVSV-MACV5 elicits higher levels of total IgG and neutralizing antibodies than rVSV-MACV1, but similar levels of MACV-specific T cells. To further investigate vaccine strategies for MACV and other NW arenaviruses such as Jun\u00edn virus (JUNV), we evaluated the humoral immune response to sequential immunization with rVSVs expressing MACV and JUNV GPCs. The highest levels of MACV and JUNV reactive antibodies were achieved using an rVSV-MACV5 prime followed by rVSV-JUNV boost. The data from our study have implications for rVSV vaccine design and provide a foundation for the development of rVSV-vectored vaccines for MACV and other NW arenaviruses.<\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Machupo virus (MACV) is a highly pathogenic New World (NW) mammarenavirus that causes Bolivian hemorrhagic fever (BHF). Because of their high mortality rates, MACV and other NW arenaviruses represent a significant public health threat. However, no approved vaccines for MACV exist. Here, we describe recombinant vesicular stomatitis virus (rVSV) vaccines that express the MACV glycoprotein complex (GPC) in different positions of the VSV genome and characterize the humoral and cellular immune response to each. We also investigate sequential immunization with rVSVs expressing MACV GPC and a related NW arenaviral antigen, Jun\u00edn virus GPC, in an effort to elicit broadly cross-reactive antibody responses. The conclusions from our study are twofold: data presented here provide further understanding of rVSV vaccine vectors and how genome rearrangements can be leveraged for vaccine development, and the results from our study may inform future vaccine development for MACV and other NW arenaviruses.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00357-26","type":"journal-article","created":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T13:00:26Z","timestamp":1784898026000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Immunogenicity of recombinant vesicular stomatitis virus-vectored vaccines expressing the Machupo virus glycoprotein in different genome positions"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8857-4583","authenticated-orcid":true,"given":"Rachel","family":"Erickson","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00b30xv10","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania","place":["Philadelphia, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3918-5976","authenticated-orcid":true,"given":"Paul","family":"Bates","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00b30xv10","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania","place":["Philadelphia, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","unstructured":"NIAID emerging infectious diseases\/ pathogens. 2018. 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