A lab-worker was infected with HIV-1 in a biosafety level-2 of containment, without any apparent breach. Through full-genome sequencing and phylogenetic analyses, we could identify the source of infection in a replication-competent clone, unknowingly contaminating a safe experiment. Mode of transmission remains unclear. Caution is warranted when handling HIV-derived constructs

A. Bandera

A. Bertoli

A. Gori

A. Soria

C. Alteri

C.F. Perno

D. Trabattoni

E. Balestra

F. Ceccherini-Silberstein

F. Continenza

G. Scarlatti

L. Carioti

M. Biasin

M. Tolazzi

M.C. Bellocchi

AIR Universita degli studi di Milano

© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. 
All rights reserved. For permissions, e-mail: journals.permissions@oup.com
    
 
1 
Occupational HIV infection in a research laboratory with unknown mode of 
transmission: a case report  
 
Alessandro Soria
1*
, Claudia Alteri
2*
, Gabriella Scarlatti
3
, Ada Bertoli
2
, Monica Tolazzi
3
, 
Emanuela Balestra
2
, Maria Concetta Bellocchi
2
, Fabio Continenza
4
, Luca Carioti
2
, Mara 
Biasin
5
, Daria Trabattoni
5
, Alessandra Bandera
1
, Francesca Ceccherini-Silberstein
2
, Carlo 
Federico Perno
2#
, Andrea Gori
1# 
 
1
Division of Infectious Diseases, San Gerardo Hospital, University of Milano-Bicocca, 
Monza, Italy 
2
Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, 
Italy 
3
Viral Evolution and Transmission Unit,
 
Division of Immunology, Transplantation and 
Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy 
4
National Institute for Infectious Diseases L. Spallanzani-IRCCS, Rome, Italy 
5Department of Biomedical and Clinical Sciences “L. Sacco”, Chair of Immunology, 
University of Milan, Milan, Italy 
 
*
These authors equally contributed to this work 
#
Co-senior authors 
 
Corresponding author: Alessandro Soria, San Gerardo Hospital, Via Pergolesi 33, 20900 
Monza (MB), Italy, Phone +39 039 233 9590 Fax +39 039 233 9327 e-mail 
a.soria@hsgerardo.org 
 
    
 
2 
Previous presentation: Preliminary results were presented by Claudia Alteri et al. as an oral 
late breaker at the Conference on Retroviruses and Opportunistic Infections, February 22-25, 
2016, Boston, Massachusetts, abstract n.18LB. 
 
    
 
3 
ABSTRACT 
A lab-worker was infected with HIV-1 in a biosafety level-2 of containment, without any 
apparent breach. Through full-genome sequencing and phylogenetic analyses, we could 
identify the source of infection in a replication-competent clone, unknowingly contaminating 
a safe experiment. Mode of transmission remains unclear. Caution is warranted when 
handling HIV-derived constructs. 
 
Keywords: HIV-1; occupational exposure; laboratory safety; HIV transmission 
 
    
 
4 
INTRODUCTION 
HIV-1-based lentiviral vectors are widely used in research laboratories to produce viral 
particles for gene-delivery, drug-screening, and HIV replication studies [1,2]. These safe, 
non-replication-competent viral particles derive from simultaneous transfection of producer 
cells with HIV-1 defective expression constructs and autologous/heterologous envelope-
encoding plasmids, such as for HIV-1 Env, or vesicular stomatitis virus glycoprotein (VSV-
G), the latter being commonly used for its pantropism [3]. Their recognized safety [4] has 
allowed handling of these non-replication-competent viruses or pseudoviruses in a biosafety 
level (BSL)-2 conditions [5,6].  
Here we describe a case of HIV-1 infection, which occurred during HIV-1 pseudoviruses 
production in a BSL-2 containment, due to unaware contamination of these procedures with a 
replication-competent recombinant clone. 
  
CASE REPORT 
A blood donor resulted HIV-1-positive at routine screening. Previous tests (the latest 7 
months before) were negative. At first clinical assessment, investigating the HIV-acquisition 
route, no classical risk factors emerged. However, during the six months preceding HIV-1 
diagnosis, the subject worked in a research laboratory, with the exclusive task of producing 
pseudoviruses by co-transfecting 293T cells with HIV-1 NL4-3 env/nef-defective, green 
fluorescein protein-labeled vector, and JRFL Env- or VSV-G-encoding plasmid in a BSL-2 
containment. No access to BSL-3 was allowed and reported. No major or minor laboratory 
accidents (percutaneous injury, medium splash, breaking of gloves, eye or skin contact with 
cell-derived medium, etc.) were recalled. 
    
 
5 
METHODS 
Ethical statement. After Institutional and Ethics Committee approval, the patient gave 
written informed consent for all the analyses and the publication of this report.   
Viro-immunological parameters. Plasma HIV-RNA and CD4+ cell count were evaluated 
every 4 months from diagnosis. Total HIV-DNA was measured at month 12 [7]. 
HIV-1 sequencing. HIV-1 from plasma and peripheral blood mononuclear cells (PBMCs) 
was sequenced and analyzed at different time-points by bulk and ultra-deep pyrosequencing 
(UDPS). 
Phylogenetic analysis. Maximum-likelihood methods (ML) [8], using the full-length HIV-
DNA sequence (month 12), were applied to define the infectious source. Viral evolution was 
evaluated through dN/dS ratio, number of Env-glycosylation sites, and co-receptor usage at 
different time-points. 
Viral isolation, phenotypic determination, and neutralizing activity assessment. Thirty-six 
months post-diagnosis the virus was isolated from patient’s PBMCs, and phenotypically 
characterized on U87.CD4+ cells expressing CCR5 and CXCR4 [9].  Patient’s neutralizing 
antibody response was assessed by testing the virus for sensitivity to patient’s sera collected 
at different time-points, and to a panel of monoclonal antibodies (MoAbs) [10].  
Immunological characterization. At month 36, cytometric analyses were performed on HIV 
peptides-stimulated and unstimulated PBMCs. 
 
Full methodology in Supplementary Text, Tables S1-S2. 
    
 
6 
RESULTS 
Viral sequence characterization. At HIV-1 diagnosis, Western blot was positive to Env, Pol, 
Gag, and low-reactive to p31. CD4+ cell count and plasma HIV-1 RNA were 577 cells/μL 
and 119 (2.1 log10) copies/mL, respectively (Figure S1).  
The full-length sequencing showed the presence of a whole HIV-1 genome. The ML 
phylogenetic trees inferred from gag-pol, vif-vpr-vpu-tat/rev (first-exon), and nef sequences 
demonstrated that in these three regions the patient’s virus formed well-supported clusters 
with NL4-3 (bootstrap: 100%, 100%, 97.8% respectively; Figure 1). The high homology with 
NL4-3 was confirmed by the extremely low genetic distance between the two strains: 
0.004±0.001 for gag-pol, 0.008±0.003 for vif-vpr-vpu-tat/rev (first-exon), and 0.006±0.003 
for nef. The few amino acid changes from NL4-3 reference sequence in accessory genes are 
shown in Table S3. 
In contrast, the ML tree obtained by the full-length env and tat/rev (second-exon) revealed 
that the patient’s virus formed a well-supported cluster with JRFL (bootstrap: 99.7%; genetic 
distance: 0.017±0.002; Figure 1).  
Although the phylogenetic results showed a high homology between the patient’s virus and 
the HIV-1 constructs he/she was supposed to have handled, the presence of nef in the 
patient’s virus suggested that the source of infection should not have been the recombination 
between HIV-1 NL4-3 env/nef-defective vector and JRFL Env-encoding plasmid, but more 
likely an unaware use (due to contamination or labeling error) of a full-length infective clone. 
A retrospective evaluation of the clones present in the laboratory at that time identified an 
HIV-1NL4-3/JRFL plasmid, with a backbone of NL4-3, and an env of JRFL, handled in the BSL-
3 of the same laboratory by other researchers for other experimental purposes. We presume 
that this plasmid erroneously but unknowingly entered in the BSL-2. 
    
 
7 
Tropism characterization. Serial plasma and PBMCs V3 UDPS showed predominant R5-
viruses; only at month 36 two minority species emerged, with a false-positive-rate (FPR) 
<10% (below the cut-off to predict R5-tropism) and the mutation S11D, at position important 
for coreceptor binding (Table S4). This mutation was also present in the virus isolated from 
patient’s PBMCs at month 36 (Figure S2). This virus exhibited an R5 phenotypic tropism in 
U87 cells, despite a FPR of 8.1% at genotypic assay, suggesting that this single mutation, in 
this particular backbone, is not probably enough to determine X4 phenotype. 
Virus sensitivity to autologous serum and monoclonal antibodies. Patient’s sera were 
unable to neutralize the patient’s primary virus, but neutralized the pseudovirus JRFL at high 
titers. The neutralization assay performed with MoAbs showed a high sensitivity of the 
patient’s virus to gp120-directed MoAbs PG9 and PG16 but less to b12, 2G12 or the gp41-
directed MoAbs 2F5 and 4E10 (Table S5). The pseudovirus JRFL showed an inverse pattern 
of neutralization sensitivity. This may suggest an escape of autologous virus through 
evolution. Indeed, env sequencing at different time-points showed a substantial C2/V3 env 
modifications (defined by the increasing dN/dS ratio, which varied from 0.00 at month 4 to 
2.00 at month 36, and by the heterogeneity of V3 quasispecies) (Table S4). Serial gag and nef 
sequencing also revealed a genetic evolution (dN/dS ratio: 0.00 and 0.01 at month 12, and 
0.22 and 0.63 at month 36, respectively), and the emergence of amino acid changes at critical 
residues for Cytotoxic-T-Lymphocyte-escape (Table S3).  
Immunological characterization. Peripheral immune activation, higher in the patient than in 
healthy controls, the skewed distribution of CD4+ and CD8+ sub-populations observed in the 
patient, and the higher percentage of TNF /IFN -producing CD8+ cells perforin and 
granzyme-expressing CD8+ cells observed after stimulation with Gag and Env predicted a 
high cytotoxic effector potential, and did not differentiate this case from typical clinical 
course of HIV infection (Table S6). 
    
 
8 
Clinical follow-up. At month 36, CD4+ cell counts fell below 400 cells/μL and plasma HIV-
RNA increased to 20,208 (4.3 log10) copies/mL. Following the guidelines of that time, 
antiretroviral therapy with tenofovir/emtricitabine/rilpivirine was started; viral suppression 
was achieved at week 10, and maintained thereafter (Figure S1). 
    
 
9 
DISCUSSION 
Here we describe for the first time an HIV-1 infection of a laboratory worker with a 
replication-competent recombinant clone, which was erroneously and inadvertently 
introduced in a BSL-2 containment during routine experiments of HIV-1 pseudoviruses 
production. The relevant novelty, which differentiates this case from the other rare laboratory 
accidents (where laboratory workers consciously had handled infectious viral cultures) [11] is 
that the patient described here was supposed to exclusively manage constructs that were per 
se non-infectious, with restrictions for BSL-2, and that an unaware contamination by a 
recombinant clone caused the infection. 
Initial viral load and CD4+ counts dynamics depicted a slow progression during the first 2.5 
years. The subsequent viro-immunological deterioration, the successful escape from host 
immune system, and the marked genetic evolution (documented with the modification in gag, 
nef and env, including the heterogeneity of V3 quasispecies) reveal that a pure laboratory 
recombinant clone has adapted to a human host encountered for the first time, thus 
determining a progressive HIV-1 infection. 
This case is disturbing for the unexplained route of virus transmission. An in-depth 
retrospective examination of the events did not recall any accident nor any type of evident 
minor exposure to the source of infection. In this respect, we wonder if VSV-G may have 
played a favouring role. It was previously demonstrated that the plasmid encoding for VSV-G 
allows to obtain pantropic viral particles able to deliver the expression construct to a wide 
range of mammalian and non-mammalian target cells [3], thus dramatically enhancing viral 
infectivity. Previous studies have demonstrated that HIV-1 produced in cells infected with 
VSV-G gives rise to phenotypically mixed virions with an expanded host range [12,13].
 
Thus, it is conceivable that if the infectious HIV-1NL4-3/JRFL plasmid, instead of the HIV-1 
NL4-3 env/nef-defective vector, was erroneously transfected with VSV-G plasmid into 
    
 
10 
producer cells, hybrid virions containing the VSV-G and the full HIV-1 genome might have 
been generated. This could have expanded viral tropism, augmented viral infectivity, and 
extremely facilitated infection, so that even unperceived droplets of supernatant of a high-
level replicating hybrid viral culture, getting inadvertently in contact with mucous 
membranes, may have caused an accidental, unaware infection.  
Despite intensive investigation, the lack of identification of the “breach-point” that led to 
contamination, and the mode of transmission that remains obscure hinder the full 
comprehension of this accident. 
Notwithstanding these limitations, this case warns of potential hazard in research laboratories 
where multiple HIV-derived constructs are managed: BSL-2 are often large spaces occupied 
by many laboratory personnel working on different lists of projects (also conducted in BSL-
3) and sharing laboratory equipment. A precise risk assessment and procedures’ control, 
applied to every research project, could contribute to minimize biohazards and prevent at best 
laboratory exposures. 
 
  
    
 
11 
NOTES: 
AUTHORS’ CONTRIBUTIONS 
A.S. & C.A. equally contributed to this work, and followed the case by clinical and scientific 
point of view. Both carried out analysis and interpretation of data and both drafted the 
manuscript. G.S. and M.T. performed viral isolation, phenotypic determination, and 
neutralizing activity assessment. A.B. (Ada Bertoli) carried out acquisition of bulk 
sequencing data. E.B. performed VSV neutralization assay. M.C.B., F.C., L.C. carried out 
ultra-deep pyrosequencing and bioinformatic analyses. M.B., D.T., A.B. (Alessandra 
Bandera) carried out experiments for immunological characterization and corresponding data 
interpretation. A.S., C.A., F.C.S., C.F.P., A.G. participated in study conception and design. 
G.S., F.C.S., C.F.P., A.G. substantially contributed to critical revision of the manuscript. All 
the authors accepted the final version of the manuscript. 
 
ACKNOWLEDGEMENTS 
Envelope plasmids of CM235.EC4, GS015.EC12, TH023.C06 were kindly obtained from Dr. 
Nelson Michael, MHRP, USA, the one of JRFL from Dr. Rogier Sanders, AMC, The 
Netherlands, those of SF162, MW965.26, DU151 from the NIBSC Center for AIDS reagents, 
UK. Monoclonal Antibodies PG9, PG16, 4E10, 2F5, b12 and 2G12 were kindly provided by 
Dr. Dietmar Katinger, Polyimmune, Austria through the EUROPRISE project. 
We thank Ilaria Beretta, Sergio Malandrin, Massimiliano Bruni, Patrizia Saccomandi, and 
Rossana Scutari for sample and data management. Finally, we are grateful to the patient for 
his/her willingness to provide detailed history on his/her personal experience and to offer 
his/her time and materials for all the analyses performed beyond clinical practice. 
 
 
    
 
12 
FUNDING 
Costs of laboratory analyses were funded through Italian Ministry of Health (CUP: 
E81J10000000001, Ricerca Corrente and Progetto AIDS grant number 40H78) and 
AVIRALIA FOUNDATION. Cost of sample management and transport was provided by 
ANLAIDS, sezione Lombardia. The funding sources had no role in data collection, data 
analysis, and data interpretation. 
 
Conflicts of interest related to this manuscript: Alessandro Soria: no conflict; Claudia 
Alteri: no conflict; Gabriella Scarlatti: no conflict; Ada Bertoli: no conflict; Monica Tolazzi: 
no conflict; Emanuela Balestra: no conflict; Maria Concetta Bellocchi: no conflict; Fabio 
Continenza: no conflict; Luca Carioti: no conflict; Mara Biasin: no conflict; Daria Trabattoni: 
no conflict; Alessandra Bandera: no conflict; Francesca Ceccherini-Silberstein: no conflict; 
Carlo Federico Perno: no conflict; Andrea Gori: no conflict. 
 
    
 
13 
REFERENCES 
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1259-1265. 
    
 
14 
10. Fenyö EM, Heath A, Dispinseri S, et al. International network for comparison of 
HIV neutralization assays: the NeutNet report. PLoS One, 2009; 4: e4505. 
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Syndr, 1990; 3: 215-219. 
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15 
FIGURE LEGENDS 
Figure 1. Phylogenetic trees based on: panel A: full-length sequences encoding for the long 
terminal repeat (LTR)-5’ and the structural proteins Gag-Pol (nt: 5,007), corresponding to nt 
89 to nt 5,096 in reference strain HXB2 (accession number: K03455.1); panel B: full-length 
sequences encoding for the regulatory proteins Vif, Vpr, Vpu, Tat/Rev (first-exon) (nt: 
1,145), corresponding to nt 5,097 to nt 6,224 in reference strain HXB2 (accession number: 
K03455.1); panel C: full-length sequences encoding for the regulatory protein Nef and the 
LTR-3’ (nt: 762), corresponding to nt 8,797 to nt 9,559 in reference strain HXB2 (accession 
number: K03455.1); panel D: full-length sequences encoding for the Env polyprotein (nt: 
2,463), and Tat/Rev (second-exon), corresponding to nt 6,225 to nt 8,796 in reference strain 
HXB2 (accession number: K03455.1).  
The Maximum Likelihood tree was inferred by using PhyML program. Only bootstrap 
supports >70% are shown. The tree was rooted using a midpoint rooting. The gray box 
defines the cluster involving the patient’s virus, identified by the number 1900. 
 


Occupational HIV infection in a research laboratory with unknown mode of transmission : a case report

Soria, A

ALTERI, CLAUDIA

Scarlatti, G

BERTOLI, ADA

Tolazzi, M

BALESTRA, EMANUELA

BELLOCCHI, MARIA CONCETTA

Continenza, F

Carioti, L

Biasin, M

Trabattoni, D

Bandera, A

CECCHERINI SILBERSTEIN, FRANCESCA

PERNO, CARLO FEDERICO

Gori, A.

Occupational HIV infection in a research laboratory with unknown mode of transmission: a case report

https://air.unimi.it/retrieve/handle/2434/477730/795474/177%20proof.pdf

Occupational HIV infection in a research laboratory with unknown mode of transmission : a case report

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