Rhesus macaques (RMs) are valuable large-animal models for studying human pathogens, providing insights into protective immunity and pre-clinical tests of vaccine efficacy. Since antibodies are the primary mediator of humoral immune responses, monoclonal antibodies (mAbs) are often isolated to investigate their specificity, function, and variable (V) gene usage. However, cloning antibodies from RMs is challenging. Although phage-display and B-cell immortalization platforms have been used to isolate mAbs from RMs (Holman et al., 2017; Kuwata et al., 2011; Samsel et al., 2023; Blasi et al., 2018), these approaches are laborious and often yield relatively few antigen-specific antibodies. Alternatively, PCR-based strategies aim to amplify heavy and light chain genes directly from individual B cells and produce the recombinant antibodies in mammalian cell lines (Magnani et al., 2017; Meng et al., 2015; Silveira et al., 2015; Sundling et al., 2014; Wiehe et al., 2014). However, the complexity of RM Ig genes hinders the cloning efficiency of these approaches.

PCR-based methods for cloning RM antibodies are built on well-established protocols for isolating mAbs from single-cell sorted human and murine B cells (Tiller et al., 2008; Wrammert et al., 2008; Guthmiller et al., 2019; von Boehmer et al., 2016). These cloning strategies use nested PCRs to amplify immunoglobulin variable (IgV) genes. In these schemes, the 5′ primers typically anneal to the Ig leader sequences, which are immediately upstream of the IgV genes and are relatively conserved within human and murine V gene families. Thus, for these species, only small panels of 5′ primers are needed to capture most IgV genes. In contrast, RM heavy and light chain genes have more copy-number variations and diversity than their human or murine orthologs (Ramesh et al., 2017; Vázquez Bernat et al., 2021; Cirelli et al., 2019). To compensate for this increased genetic diversity, large cocktails of multiplex (MTPX) primers are used to amplify RM IgV genes (Sundling et al., 2012b; Silveira et al., 2015; Mason et al., 2016; Wiehe et al., 2014; Feng et al., 2023). However, increasing the number of primers can reduce PCR efficiency through competition for template binding and inter-primer interference. Moreover, although recent studies have advanced our understanding of the RM Ig loci (Cirelli et al., 2019; Vázquez Bernat et al., 2021; Chernyshev et al., 2021; Ramesh et al., 2017), gaps in the RM Ig gene repertoire likely remain. Thus, existing 5′ MTPX primer panels may be incomplete and unable to capture all of the RM IgV genes.

Antigen-specific memory B cells are often used as source material for cloning antibodies. These antigen-experienced cells express ample B-cell receptors (BCRs), allowing the use of fluorescently-labeled antigenic probes to select individual antigen-binding B cells via fluorescent-activated cell sorting (FACS). However, since memory B cells are not actively secreting antibodies, they have a low abundance of antibody transcripts (Phad et al., 2022; Meng et al., 2015; Tiller et al., 2008; Upadhyay et al., 2018), limiting the recovery of heavy and light chain pairs (Meng et al., 2015).

The first step in cloning antibodies from individual memory B cells using PCR-based methods is converting mRNA into cDNA using reverse transcriptase (RT). SMART (switching mechanism at the 5′ ends of the RNA transcript) technology, a refinement of 5′ and 3′ rapid amplification of cDNA ends (5′/3′ RACE) synthesis, is a useful technique for generating full-length cDNA libraries when mRNA transcripts are low (Zhu et al., 2001). SMART technology takes advantage of the natural poly(A) tail of mRNAs and the template-switching activity of Moloney murine leukemia virus (MMLV)-based RTs. During 3′ RACE reactions, cDNA synthesis is initiated using primers containing deoxythymidine (dT) tracts that are complementary to mRNA poly(A) tails. As RTs with transferase activity reach the 5′ cap of mRNAs, they add non-templated nucleotides, predominantly deoxycytidines (+CCC), to the 3′ end of nascent cDNA molecules (Kulpa et al., 1997). The newly added +CCC bases can then bind template-switching oligos (TSOs) with complementary riboguanosine nucleotides (rGrGrG) at their 3′ end (Zhu et al., 2001). This prompts the RT to complete cDNA synthesis using the TSO as the template, effectively switching templates in the process. 5′ RACE is particularly useful when the 5′ ends of transcripts are highly variable and designing primers for downstream amplifications is challenging. In such cases, synthetic primer binding sites can be incorporated into the TSOs and added to the 5′ ends of cDNAs during synthesis. As a result, 5′ universal primers complementary to the primer binding sites can be paired with gene-specific 3′ primers in PCRs to amplify target genes (Zhu et al., 2001).

In this study, we present a novel method for isolating mAbs from individual RM memory B cells. One of the key features of this protocol is the incorporation of synthetic primer binding sites to the 5′ and 3′ ends of cDNAs during synthesis. This approach offers several advantages for rescuing antibodies. First, low-abundance templates can be PCR amplified using 5′ and 3′ universal primers, which yields sufficient material for downstream PCRs. Second, appending two synthetic primer binding sites into the 5′ end of the cDNAs enables using 5′ universal primers in nested PCR reactions, eliminating the need for complex 5′ MTPX primer sets to amplify the IgV genes. We demonstrate this process by isolating mAbs specific for the envelope (Env) protein of simian immunodeficiency virus (SIV) from single-cell sorted rhesus macaque memory B cells. We anticipate this method will improve the recovery of monoclonal antibodies from RMs and facilitate the characterization of antigen-specific B cells in pre-clinical studies.